Method for sample preservation and nucleic acid release, and method for nucleic acid detection

Abstract

Provided are a formulation for enhancing nucleic acid release efficiency in a sample and a use thereof. Specifically, the formulation comprises a preservation solution, and further comprises grinding filter beads. Using the formulation of the present application to treat a sample can enhance the nucleic acid release efficiency of the sample. With the assistance of ultrasound and / or heating, the nucleic acid release efficiency of the sample is further improved, thereby simplifying sample collection and processing procedures.

Description

A method for releasing, preserving and detecting nucleic acid from samples Technical Field

[0001] This application relates to the field of sample detection technology, specifically to a method for releasing, preserving, and detecting nucleic acid in samples. Background Technology

[0002] Respiratory infections are among the most common illnesses people contract in daily life. The pathogens causing these infections are typically highly contagious, leading most hospitals to establish dedicated fever clinics. Routine molecular testing requires nucleic acid extraction followed by PCR testing, a process that usually takes about two hours. This extended waiting time for feverish patients increases the risk of pathogen transmission or cross-infection.

[0003] To address the aforementioned issues, those skilled in the art have developed cartridge-based POCT testing instruments and reagents. Compared to the traditional magnetic bead method, cartridge-based reagents can shorten the overall testing time and reduce patient waiting time. However, the complex manufacturing process of cartridge-based reagents leads to higher reagent costs, making the high testing fees prohibitive for many patients. Furthermore, the quality of cartridge-based reagents cannot be fully controlled, as it is impossible to test every consumable; if a testing problem occurs, resampling may be necessary.

[0004] A direct amplification method using a release agent without nucleic acid extraction was also developed. This method involves immersing swab samples in a special release agent liquid to release nucleic acids. However, this method is prone to false negatives. The main reasons for this are likely that the method releases nucleic acids while lysing pathogens, leading to poor nucleic acid stability; additionally, RNA may be degraded before detection, resulting in false positives; and because this method does not involve nucleic acid purification, inhibitors collected from the patient's mouth, such as food residue, can easily be introduced into the detection system, inhibiting the amplification reaction.

[0005] Therefore, there is an urgent need in this field for an efficient, pollution-free, and non-destructive method for sample preservation and nucleic acid release. Summary of the Invention

[0006] This application provides an efficient, pollution-free, and non-destructive method for sample preservation and nucleic acid release, and also provides an efficient, pollution-free, and non-destructive sample preservation and nucleic acid release solution.

[0007] In a first aspect, this application provides a formulation for sample preservation and nucleic acid release, the formulation comprising a preservation solution, the preservation solution being composed of a release component, a nucleic acid stabilizing component, and a buffer component;

[0008] The releasing component consists of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant.

[0009] The nucleic acid stabilizing component consists of 0.01%–10% (w / v) nucleic acid protectant and 0.1–20 mM metal ion chelating agent;

[0010] The buffer component is 0.1–50 mM of tris(hydroxymethyl)aminomethane (Tris);

[0011] The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof.

[0012] The nonionic surfactant is selected from the group consisting of: nonylphenol polyoxyethylene ether (NP-40), polysorbate-20 (Tween-20), polyethylene glycol octylphenyl ether (Triton-X-100), polyoxyethylene lauroyl ether (Brij-35), or combinations thereof;

[0013] The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof;

[0014] The metal ion chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), or combinations thereof.

[0015] In one embodiment, the preservation solution consists of 0.01%–5% (w / v) SDS, 0.01%–5% (w / v) NP-40, 0.01%–10% (w / v) trehalose, 0.1–20 mM EDTA, and 0.1–50 mM Tris.

[0016] In one embodiment, the preservation solution consists of 0.01%–3% (w / v) SDS, 0.02%–3% (w / v) NP-40, 0.05%–5% (w / v) trehalose, 1–10 mM EDTA and 1–30 mM Tris.

[0017] In one embodiment, the preservation solution consists of 0.01%–1% (w / v) SDS, 0.02%–2% (w / v) NP-40, 0.1%–3% (w / v) trehalose, 3–8 mM EDTA, and 10–20 mM Tris.

[0018] In one embodiment, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0019] In one embodiment, the preservation solution contains no protein and no enzymes.

[0020] In one embodiment, the formulation further includes milled filter beads.

[0021] In one embodiment, the grinding filter beads are modified with hydroxyl or carboxyl groups.

[0022] In one embodiment, the abrasive filter beads are selected from the group consisting of: quartz sand, zirconium oxide beads, foam filter beads, activated carbon particles, bio-ceramic particles, molecular sieves, fiber balls, or combinations thereof; preferably, bio-ceramic particles and molecular sieves.

[0023] In one embodiment, the concentration of the abrasive filter beads in the formulation or the preservation solution is 0.01–5 g / mL, preferably 0.05–3 g / mL, more preferably 0.1–2 g / mL, and most preferably 0.2–1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL, or 1 g / mL.

[0024] In one embodiment, the pH of the preservation solution is 7 to 12, preferably 8 to 12, more preferably 9 to 12, and most preferably 10 to 12, for example, about 11.

[0025] Secondly, this application provides a kit for sample preservation and nucleic acid release, the kit comprising:

[0026] A first container and a preservation solution located in the first container, the preservation solution being composed of a release component, a nucleic acid stabilizing component, and a buffer component;

[0027] The releasing component consists of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant.

[0028] The nucleic acid stabilizing component consists of 0.01%–10% (w / v) nucleic acid protectant and 0.1–20 mM metal ion chelating agent;

[0029] The buffer component is 0.1–50 mM Tris;

[0030] The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof.

[0031] The nonionic surfactant is selected from the group consisting of: NP-40, Tween-20, Triton-X-100, Brij-35, or combinations thereof;

[0032] The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof;

[0033] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof.

[0034] In one embodiment, the kit further includes grinding filter beads.

[0035] In one embodiment, the preservation solution and the grinding filter beads are located in the same or different containers.

[0036] In one embodiment, the grinding filter beads are modified with hydroxyl or carboxyl groups.

[0037] In one embodiment, the abrasive filter beads are selected from the group consisting of: quartz sand, zirconium oxide beads, foam filter beads, activated carbon particles, bioceramic granules, molecular sieves, fiber balls, or combinations thereof.

[0038] In one embodiment, the preservation solution consists of 0.01%–5% (w / v) SDS, 0.01%–5% (w / v) NP-40, 0.01%–10% (w / v) trehalose, 0.1–20 mM EDTA and 0.1–50 mM Tris.

[0039] In one embodiment, the preservation solution consists of 0.01%–3% (w / v) SDS, 0.02%–3% (w / v) NP-40, 0.05%–5% (w / v) trehalose, 1–10 mM EDTA, and 1–30 mM Tris.

[0040] Preferably, the preservation solution consists of 0.01%–1% (w / v) SDS, 0.02%–2% (w / v) NP-40, 0.1%–3% (w / v) trehalose, 3–8 mM EDTA and 10–20 mM Tris;

[0041] More preferably, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0042] In one embodiment, when the preservation solution and the grinding filter beads are located in the same container, the concentration of the grinding filter beads in the preservation solution is 0.01 to 5 g / mL, preferably 0.05 to 3 g / mL, more preferably 0.1 to 2 g / mL, and most preferably 0.2 to 1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL or 1 g / mL.

[0043] In one embodiment, the pH of the preservation solution is 7 to 12, preferably 8 to 12, more preferably 9 to 12, and most preferably 10 to 12, for example, about 11.

[0044] In one embodiment, the preservation solution contains no protein and no enzymes.

[0045] In one embodiment, the kit further includes an instruction manual specifying that the kit is used for sample preservation and nucleic acid release, and also specifying the method of use for sample preservation and nucleic acid release.

[0046] Thirdly, this application provides a method for increasing the nucleic acid release rate in a sample, comprising the following steps:

[0047] (S1) Provide a sample to be tested;

[0048] (S2) Add the sample to the preservation solution and mix well to preserve the sample and improve the nucleic acid release rate in the sample;

[0049] The preservation solution consists of a release component, a nucleic acid stabilizing component, and a buffer component;

[0050] The releasing component consists of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant.

[0051] The nucleic acid stabilizing component consists of 0.01%–10% (w / v) nucleic acid protectant and 0.1–20 mM metal ion chelating agent;

[0052] The buffer component is 0.1–50 mM Tris;

[0053] The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof.

[0054] The nonionic surfactant is selected from the group consisting of: NP-40, Tween-20, Triton-X-100, Brij-35, or combinations thereof;

[0055] The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof;

[0056] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof.

[0057] In one embodiment, the method further includes step (S3) of adding grinding filter beads to the preservation solution containing the sample and mixing them.

[0058] In one embodiment, the grinding filter beads are modified with hydroxyl or carboxyl groups;

[0059] In one embodiment, the abrasive filter beads are selected from the group consisting of: quartz sand, zirconium oxide beads, foam filter beads, activated carbon particles, bioceramic granules, molecular sieves, fiber balls, or combinations thereof.

[0060] In one embodiment, the method further includes step (S4) of sonicating and / or heating the preservation solution containing the sample.

[0061] In one embodiment, the sample-containing preservation solution further contains abrasive filter beads.

[0062] In one embodiment, the preservation solution consists of 0.01%–5% (w / v) SDS, 0.01%–5% (w / v) NP-40, 0.01%–10% (w / v) trehalose, 0.1–20 mM EDTA and 0.1–50 mM Tris.

[0063] In one embodiment, the preservation solution consists of 0.01%–3% (w / v) SDS, 0.02%–3% (w / v) NP-40, 0.05%–5% (w / v) trehalose, 1–10 mM EDTA and 1–30 mM Tris.

[0064] In one embodiment, the preservation solution consists of 0.01%–1% (w / v) SDS, 0.02%–2% (w / v) NP-40, 0.1%–3% (w / v) trehalose, 3–8 mM EDTA and 10–20 mM Tris.

[0065] In one embodiment, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0066] In one embodiment, the pH of the preservation solution is 7 to 12, preferably 8 to 12, more preferably 9 to 12, and most preferably 10 to 12, for example, about 11.

[0067] In one embodiment, in step (S3), 0.01 to 5 g of grinding filter beads are added to every 1 mL of preservation solution.

[0068] In one embodiment, in step (S3), 0.05 to 3 g of grinding filter beads are added to every 1 mL of preservation solution, preferably 0.1 to 2 g, more preferably 0.2 to 1 g, for example, about 0.2 g, 0.4 g, 0.6 g, 0.8 g or 1 g.

[0069] In one embodiment, the sample is an oropharyngeal swab and / or a nasopharyngeal swab.

[0070] In one embodiment, the sample contains pathogens, preferably viruses, bacteria and / or mycoplasma, more preferably influenza A virus, adenovirus, mycoplasma pneumoniae, human metapneumovirus, or combinations thereof.

[0071] In one embodiment, the preservation solution contains no protein and no enzymes.

[0072] In one embodiment, in step (S4), the ultrasonic treatment and the heating treatment are performed simultaneously or separately.

[0073] In one embodiment, in step (S4), the frequency of the ultrasonic treatment is 30-50 kHz, preferably 35-45 kHz, and more preferably 35-40 kHz.

[0074] In one embodiment, in step (S4), the ultrasonic treatment lasts for 30 to 240 seconds, preferably 30 to 180 seconds, and more preferably 120 to 180 seconds.

[0075] In one embodiment, in step (S4), the heating treatment is heating at 75-95°C for 1-5 minutes, preferably heating at 75-95°C for 1-3 minutes.

[0076] In one embodiment, the nucleic acid release rate of the method is ≥85%, preferably ≥90%, and more preferably ≥95%.

[0077] Fourthly, this application provides a nucleic acid detection method, which includes the following steps:

[0078] (z1) Add the sample to be tested into the preservation solution and mix well. The preservation solution consists of a release component, a nucleic acid stabilizing component and a buffer component.

[0079] The releasing component consists of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant.

[0080] The nucleic acid stabilizing component consists of 0.01%–10% (w / v) nucleic acid protectant and 0.1–20 mM metal ion chelating agent;

[0081] The buffer component is 0.1–50 mM Tris;

[0082] The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof.

[0083] The nonionic surfactant is selected from the group consisting of: NP-40, Tween-20, Triton-X-100, Brij-35, or combinations thereof;

[0084] The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof;

[0085] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof; and

[0086] (z2) Nucleic acid detection is performed on the preservation solution containing the sample to be tested to obtain the nucleic acid detection result.

[0087] In one embodiment, step (z3) is further included between steps (z1) and (z2): adding grinding filter beads to the preservation solution containing the sample to be tested and mixing them.

[0088] In one embodiment, in step (z2), the preservation solution contains abrasive filter beads and the sample to be tested.

[0089] In one embodiment, step (z4) is further included between steps (z1) and (z2) to sonicate and / or heat the preservation solution containing the sample to be tested.

[0090] In one implementation, steps (z3) and (z4) are included between steps (z1) and (z2).

[0091] In one implementation, step (z4) is performed after step (z3).

[0092] In one embodiment, in step (z4), the preservation solution contains grinding filter beads and the sample to be tested.

[0093] In one embodiment, in step (z2), the preservation solution is diluted 1 to 20 times, preferably 5 to 15 times, more preferably 8 to 12 times, for example, about 10 times, before nucleic acid detection.

[0094] In one embodiment, the grinding filter beads are modified with hydroxyl or carboxyl groups.

[0095] In one embodiment, the abrasive filter beads are selected from the group consisting of: quartz sand, zirconium oxide beads, foam filter beads, activated carbon particles, bioceramic granules, molecular sieves, fiber balls, or combinations thereof.

[0096] In one embodiment, the anionic surfactant is sodium dodecyl sulfate; the nonionic surfactant is NP-40.

[0097] In one embodiment, the preservation solution consists of 0.01%–5% (w / v) SDS, 0.01%–5% (w / v) NP-40, 0.01%–10% (w / v) trehalose, 0.1–20 mM EDTA and 0.1–50 mM Tris.

[0098] In one embodiment, the preservation solution consists of 0.01%–3% (w / v) SDS, 0.02%–3% (w / v) NP-40, 0.05%–5% (w / v) trehalose, 1–10 mM EDTA and 1–30 mM Tris.

[0099] In one embodiment, the preservation solution consists of 0.01%–1% (w / v) SDS, 0.02%–2% (w / v) NP-40, 0.1%–3% (w / v) trehalose, 3–8 mM EDTA and 10–20 mM Tris.

[0100] In one embodiment, the preservation solution consists of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0101] In one embodiment, the pH of the preservation solution is 7 to 12, preferably 8 to 12, more preferably 9 to 12, and most preferably 10 to 12, for example, about 11.

[0102] In one embodiment, in step (z3), 0.01 to 5 g of grinding filter beads are added to every 1 mL of preservation solution.

[0103] In one embodiment, in step (z3), 0.05 to 3 g of grinding filter beads are added to every 1 mL of preservation solution, preferably 0.1 to 2 g, more preferably 0.2 to 1 g, for example, about 0.2 g, 0.4 g, 0.6 g, 0.8 g or 1 g.

[0104] In one embodiment, the sample to be tested is an oropharyngeal swab and / or a nasopharyngeal swab.

[0105] In one embodiment, the sample to be tested contains pathogens, preferably viruses, bacteria and / or mycoplasma, more preferably influenza A virus, adenovirus, mycoplasma pneumoniae, human metapneumovirus, or combinations thereof.

[0106] In one embodiment, the preservation solution contains no protein and no enzymes.

[0107] In one embodiment, in step (z4), the ultrasonic treatment and the heating treatment are performed simultaneously or separately.

[0108] In one embodiment, in step (z4), the frequency of the ultrasonic treatment is 30-50 kHz, preferably 35-45 kHz, and more preferably 35-40 kHz.

[0109] In one embodiment, in step (z4), the ultrasonic treatment lasts for 30–240 s, preferably 30–180 s, and more preferably 120–180 s.

[0110] In one embodiment, in step (z4), the heat treatment is heating at 75-95°C for 1-5 minutes, preferably heating at 75-95°C for 1-3 minutes.

[0111] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0112] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors have unexpectedly discovered for the first time a formulation combining sample preservation and processing. This formulation includes a preservation solution and further comprises grinding filter beads. The inventors optimized the components and concentration of the preservation solution through extensive experiments, thereby improving the nucleic acid release rate of the samples. Experiments show that, with the assistance of ultrasound and / or heating, the formulation of this application can further improve the nucleic acid release rate of the samples, thus simplifying the sample collection and processing procedures. The method of processing samples using the formulation of this application is simple to operate, low in cost, fast, convenient, and has a wide range of applications. Based on this, this application was completed.

[0113] the term

[0114] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Before describing this application, it should be understood that this application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of this application will be limited only by the appended claims.

[0115] As used herein, the term “comprising” or its variations such as “including” or “comprises” are understood to include the said element or component without excluding other elements or other components.

[0116] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending 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.).

[0117] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0118] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0119] As used herein, the terms “SDS” and “sodium dodecyl sulfate” are used interchangeably.

[0120] As used in this article, the terms “Tween-20” and “Tween 20” are used interchangeably.

[0121] As used herein, the terms “Triton-X-100” and “Triton X-100” are used interchangeably.

[0122] As used in this article, the term "Brij-35" refers to an ether compound formed by the reaction of lauryl alcohol and ethylene oxide. It is a nonionic surfactant with good emulsifying, wetting, dispersing and solubilizing properties.

[0123] As used herein, the terms “NP-40” and “nonylphenol polyoxyethylene ether” are used interchangeably.

[0124] As used herein, the terms “EDTA” and “ethylenediaminetetraacetic acid” are used interchangeably.

[0125] As used herein, the terms “EGTA” and “ethylene glycol tetraacetic acid” are used interchangeably.

[0126] Magnetic bead method

[0127] The magnetic bead extraction method requires releasing the pathogen's nucleic acid from the pathogen and then adsorbing it onto magnetic beads. The adsorbed nucleic acid beads are then transferred to a washing solution containing salt to further remove protein impurities and lysis buffer. After the first wash, the beads are washed a second time with a salt-free alcohol solution. The washed beads are then air-dried to remove any residual ethanol. Finally, the dried beads are transferred to a low-salt elution buffer for elution. Each step takes at least 2-5 minutes and requires the use of magnets or similar devices. If using an instrument, specific extraction equipment is required, and the entire process takes between 15 and 30 minutes.

[0128] The formulation of this application

[0129] The formulation of this application is used for sample preservation and nucleic acid release, including a preservation solution, which is composed of a release component, a nucleic acid stabilizing component and a buffer component;

[0130] The releasing component consists of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant.

[0131] The nucleic acid stabilizing component consists of 0.01%–10% (w / v) nucleic acid protectant and 0.1–20 mM metal ion chelating agent;

[0132] The buffer component is 0.1–50 mM Tris;

[0133] The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof.

[0134] The nonionic surfactant is selected from the group consisting of: NP-40, Tween-20, Triton-X-100, Brij-35, or combinations thereof;

[0135] The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof;

[0136] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof.

[0137] In the preservation solution of this application, the combination of ionic and nonionic surfactants can reduce the pressure on the system and enable the reaction system to tolerate higher concentrations of ionic surfactants. The preservation solution of this application includes nucleic acid protectants and metal chelating agents; the combination of these two can further improve the amplification system's tolerance to anionic surfactants.

[0138] In a preferred embodiment, the preservative solution comprises 0.01%–5% (w / v) SDS, 0.01%–5% (w / v) NP-40, 0.01%–10% (w / v) trehalose, 0.1–20 mM EDTA, and 0.1–50 mM Tris; preferably, the preservative solution comprises 0.01%–3% (w / v) SDS, 0.02%–3% (w / v) NP-40, 0.05%–5% (w / v) trehalose, 1–10 mM EDTA, and 1–30 mM Tris; more preferably, the preservative solution comprises 0.01%–1% (w / v) SDS, 0.02%–2% (w / v) NP-40, 0.1%–3% (w / v) trehalose, 3–8 mM EDTA, and 10–20 mM Tris. The solution is composed of Tris; most preferably, the preservation solution is composed of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0139] In a preferred embodiment, the pH of the preservation solution is 7–12, preferably 8–12, more preferably 9–12, and most preferably 10–12, for example, about 11. The amplification effect of samples treated with the preservation solution of this application is optimal at a pH of 11.

[0140] Samples preserved using the preservation solution described in this application remain stable for RNA nucleic acids at room temperature for 6 hours and at 2–8°C for 6 days.

[0141] In a preferred embodiment, the formulation further includes grinding filter beads. In a preferred embodiment, the grinding filter beads are modified with hydroxyl or carboxyl groups. In a preferred embodiment, the grinding filter beads are selected from the group consisting of: quartz sand, zirconia beads, foam filter beads, activated carbon particles, bio-ceramic particles, molecular sieves, fiber balls, or combinations thereof; preferably, bio-ceramic particles and molecular sieves. Different grinding filter beads are selected according to different application scenarios. The formulation of this application contains grinding filter beads, which can effectively remove impurities from the sample and improve nucleic acid release efficiency through physical collision. The grinding filter beads can not only assist nucleic acid release but also adsorb impurities in the sample and reduce inhibitors in the system.

[0142] In a preferred embodiment, the concentration of the abrasive filter beads is 0.01–5 g / mL, preferably 0.05–3 g / mL, more preferably 0.1–2 g / mL, and most preferably 0.2–1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL, or 1 g / mL.

[0143] The method of this application

[0144] The method of this application is used to improve the nucleic acid release rate in samples, including the following steps:

[0145] (S1) Provide a sample to be tested;

[0146] (S2) Add the sample to the preservation solution and mix well. The preservation solution consists of a release component, a nucleic acid stabilizing component and a buffer component.

[0147] The releasing component consists of 0.01% to 5% (w / v) anionic surfactant and 0.01% to 5% (w / v) nonionic surfactant.

[0148] The nucleic acid stabilizing component consists of 0.01%–10% (w / v) nucleic acid protectant and 0.1–20 mM metal ion chelating agent;

[0149] The buffer component is 0.1–50 mM Tris;

[0150] The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof.

[0151] The nonionic surfactant is selected from the group consisting of: NP-40, Tween-20, Triton-X-100, Brij-35, or combinations thereof;

[0152] The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof;

[0153] The metal ion chelating agent is selected from the group consisting of EDTA, EGTA, or combinations thereof.

[0154] In a preferred embodiment, the method further includes step (S3) of adding grinding filter beads to the preservation solution containing the sample and mixing them.

[0155] In a preferred embodiment, the preservative solution comprises 0.01%–5% (w / v) SDS, 0.01%–5% (w / v) NP-40, 0.01%–10% (w / v) trehalose, 0.1–20 mM EDTA, and 0.1–50 mM Tris; preferably, the preservative solution comprises 0.01%–3% (w / v) SDS, 0.02%–3% (w / v) NP-40, 0.05%–5% (w / v) trehalose, 1–10 mM EDTA, and 1–30 mM Tris; more preferably, the preservative solution comprises 0.01%–1% (w / v) SDS, 0.02%–2% (w / v) NP-40, 0.1%–3% (w / v) trehalose, 3–8 mM EDTA, and 10–20 mM Tris. The preservation solution is composed of Tris; more preferably, the preservation solution is composed of 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 5 mM EDTA, and 5 mM Tris.

[0156] In a preferred embodiment, the formulation further includes abrasive filter beads. In a preferred embodiment, the abrasive filter beads are modified with hydroxyl or carboxyl groups. In a preferred embodiment, the abrasive filter beads are selected from the group consisting of: quartz sand, zirconia beads, foam filter beads, activated carbon particles, bio-ceramic granules, molecular sieves, fiber balls, or combinations thereof; preferably, bio-ceramic granules and molecular sieves.

[0157] In a preferred embodiment, the concentration of the abrasive filter beads is 0.01–5 g / mL, preferably 0.05–3 g / mL, more preferably 0.1–2 g / mL, and most preferably 0.2–1 g / mL, for example, about 0.2 g / mL, 0.4 g / mL, 0.6 g / mL, 0.8 g / mL, or 1 g / mL.

[0158] In a preferred embodiment, the method further includes step (S4) of sonicating and / or heating the preservation solution containing the sample. In one embodiment, in step (S4), the sonication and heating are performed simultaneously or separately. In a preferred embodiment, in step (S4), the frequency of the sonication is 30–50 kHz, preferably 35–45 kHz, more preferably 35–40 kHz. In a preferred embodiment, in step (S4), the sonication is performed for 30–240 s, preferably 30–180 s, more preferably 120–180 s. In a preferred embodiment, in step (S4), the heating is performed at 75–95 °C for 1–5 min, preferably 75–95 °C for 1–3 min. Sonication and / or heating can further improve the nucleic acid release rate.

[0159] In a preferred embodiment, the sample is an oropharyngeal swab and / or a nasopharyngeal swab. In a preferred embodiment, the sample contains pathogens, preferably viruses, bacteria, and / or mycoplasma, more preferably influenza A virus, adenovirus, Mycoplasma pneumoniae, human metapneumovirus, or a combination thereof.

[0160] The main advantages of this application include:

[0161] 1. The method of this application combines the use of grinding beads to assist in physical fragmentation while adsorbing impurities in the system, achieving a purification effect, reducing the inhibitors introduced into the amplification system, and effectively reducing the occurrence of false-negative samples.

[0162] 2. The method of this application combines the steps of sample collection and nucleic acid processing, effectively shortening the detection time of respiratory pathogens in the fever clinic, and shortening the entire detection process by about 20 - 30 minutes.

[0163] 3. The method of this application allows for on-demand testing, increasing the turnover efficiency of testing and reducing the waiting time of patients.

[0164] 4. The method of this application is combined with physical methods such as ultrasound or heating, which can further improve the release efficiency of sample nucleic acids and denature the inhibitors in the collected samples. With the assistance of grinding beads and physical methods, the reliability of the test results of the samples processed by this method is continuously improved.

[0165] 5. The method of this application does not require additional sophisticated instruments and consumables with complex structures, reducing the testing cost.

[0166] The following further elaborates on this application in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate this application and not to limit the scope of this application. The experimental methods without specific conditions noted in the following embodiments are usually carried out 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.

[0167] Example 1

[0168] The pseudovirus particles of influenza A virus were treated with nuclease before use. To study the sample release conditions, different surfactants were tested. Therefore, in this application, after diluting the pseudovirus with sample preservation solutions of different compositions, a portion of the pseudovirus was diluted with water by the same multiple, and after extraction using a nucleic acid extraction and purification reagent (Shanghai Feng Medical Equipment Preparation No. 20180202), it was used as a control.

[0169] Different types of surfactants were selected, and different preservation solutions were prepared according to Table 1.

[0170] Table 1

[0171] The specific operating procedure is as follows:

[0172] (1) Sample processing

[0173] Take a pseudovirus of influenza A virus and treat it with nuclease to ensure that the free nucleic acid inside is completely digested, leaving only intact virus particles.

[0174] (2) Sample release and nucleic acid extraction

[0175] The nuclease-digested pseudovirus was diluted 20-fold with the prepared preservation solution. Simultaneously, two portions of the pseudovirus were diluted with DEPC-treated water, one at a 20-fold dilution and the other at a 40-fold dilution. The preservation solution containing the pseudovirus was thoroughly mixed and incubated at room temperature for 5 minutes. The pseudovirus treated with DEPC water was then extracted using a magnetic bead-based nucleic acid extraction and purification reagent.

[0176] (3) Sample testing

[0177] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method). To prevent excessive surfactant concentration, the samples in the above preservation solution were diluted 10 times with water before simultaneous testing.

[0178] The results are shown in Table 2.

[0179] Table 2

[0180] The results of diluting the processed samples 10 times are shown in Table 3.

[0181] Table 3

[0182] Based on the above results, different surfactants all exhibited a certain nucleic acid release capacity relative to DEPC-treated water. Ionic surfactants showed better release than non-ionic surfactants, and amplification after dilution revealed that within a certain range, higher concentrations resulted in better release. However, excessively high concentrations of ionic surfactants may affect the amplification efficiency of the PCR reaction.

[0183] Based on the fact that the release effects of different anionic surfactants were basically consistent in the above experiments, sodium dodecyl sulfate was selected as the main surfactant for subsequent research based on its availability.

[0184] Example 2

[0185] Sodium dodecyl sulfate was used in combination with different nonionic surfactants to test its release capacity. Specific formulations are shown in Table 4.

[0186] Table 4

[0187] The specific operating procedure is as follows:

[0188] (1) Sample processing

[0189] Take a pseudovirus of influenza A virus and treat it with nuclease to ensure that the free nucleic acid inside is completely digested, leaving only intact virus particles.

[0190] (2) Sample release and nucleic acid extraction

[0191] The nuclease-digested pseudovirus was diluted 20-fold with the prepared preservation solution. Simultaneously, two portions of the pseudovirus were diluted with DEPC-treated water, one at a 20-fold dilution and the other at a 40-fold dilution. The preservation solution containing the pseudovirus was thoroughly mixed and incubated at room temperature for 5 minutes. The pseudovirus treated with DEPC water was then extracted using a magnetic bead-based nucleic acid extraction and purification reagent.

[0192] (3) Sample testing

[0193] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method). To prevent excessive surfactant concentration, the samples in the above preservation solution were diluted 10 times with water before simultaneous testing.

[0194] The results are shown in Tables 5 and 6.

[0195] Table 5

[0196] The test results after the samples were diluted 10 times are shown in Table 6.

[0197] Table 6

[0198] Based on the above results, the effects of using nonionic and anionic surfactants together are basically the same. Furthermore, with the addition of nonionic surfactants, the concentration of anionic surfactants that previously could not detect nucleic acid release (such as 0.10% sodium dodecyl sulfate in Table 2) can now detect nucleic acid release. Subsequent experiments will use NP-40 for further exploration.

[0199] Example 3

[0200] Surfactants can denature proteins, therefore they are also a type of nucleic acid protectant. Since they are mostly used in oropharyngeal swabs, the samples may contain various impurities from the oral cavity that could degrade nucleic acids, especially RNA. An RNA protectant was added to the components containing lysis function, with a concentration of 2×. Twenty oropharyngeal swabs were collected and thoroughly mixed in 20 mL of DEPC-treated water.

[0201] The prepared 2× solution was mixed with DEPC-treated water from the collected swabs at a 1:1 ratio and then used to dilute the extracted pseudovirus nucleic acid.

[0202] In this embodiment, the basic formulation used is 0.1% SDS and 0.2% NP-40, and the specific experimental settings are further set up on this basic formulation as shown in Table 7.

[0203] Table 7

[0204] The specific operating procedure is as follows:

[0205] (1) Sample processing

[0206] The pseudovirus of influenza A was collected and extracted using a nucleic acid extraction reagent to obtain the nucleic acid of the pseudovirus of influenza A.

[0207] Take 20 negative swabs collected from different individuals, add 20 mL of DEPC-treated water, mix thoroughly, and set aside.

[0208] (2) Sample release and nucleic acid extraction

[0209] Dilute the prepared H1N1 pseudovirus nucleic acid 5 times using the 2× concentration formulation described above, and then dilute it 2 times with a mixture of DEPC-treated negative swabs. Simultaneously, dilute the nucleic acid 10 times with DEPC-treated water. Thoroughly mix the preservation solution containing the pseudovirus nucleic acid and incubate at room temperature for 30 minutes. For pseudovirus nucleic acid treated with DEPC water, follow the same procedures as above to ensure consistent dilution.

[0210] (3) Sample testing

[0211] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0212] The results are shown in Table 8.

[0213] Table 8

[0214] Based on the above results, the addition of formamide and trehalose has a relatively small inhibitory effect on the detection system, which can ensure effective amplification.

[0215] Example 4

[0216] Further research was conducted on the combination of formamide and trehalose with metal ion chelators. RNA protectants were added to the aforementioned components containing cleavage function at a concentration of 2×. Twenty oropharyngeal swabs were collected and thoroughly mixed in 20 mL of DEPC-treated water.

[0217] The prepared 2× solution was mixed with DEPC-treated water from the collected swabs at a 1:1 ratio and then used to dilute the extracted pseudovirus nucleic acid.

[0218] In this embodiment, the basic formulation used is 0.1% SDS and 0.2% NP-40, and the specific experimental settings are further set up on this basic formulation as shown in Table 9.

[0219] Table 9

[0220] The specific operating procedure is as follows:

[0221] (4) Sample processing

[0222] The pseudovirus of influenza A was collected and extracted using a nucleic acid extraction reagent to obtain the nucleic acid of the pseudovirus of influenza A.

[0223] Take 24 negative swabs collected from different individuals, add 24 mL of DEPC-treated water, mix thoroughly, and set aside.

[0224] (5) Sample release and nucleic acid extraction

[0225] The prepared H1N1 pseudovirus nucleic acid was diluted 5-fold using the 2× concentration formulation described above, and then diluted 2-fold using DEPC-treated negative swabs. Simultaneously, the pseudovirus was diluted 10-fold with DEPC-treated water. The preservation solution containing the added pseudovirus nucleic acid was thoroughly mixed and incubated at room temperature for 30 minutes. The DEPC-treated pseudovirus nucleic acid was prepared using the same procedure as above, ensuring consistent dilution. Each sample was divided into eight portions. Four portions were incubated at room temperature for testing at 0, 3, 6, and 9 hours; the other four portions were incubated at 2-8℃ for testing at 0, 3, 6, and 9 days.

[0226] (6) Sample testing

[0227] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0228] The Ct values ​​are shown in Table 10.

[0229] Table 10

[0230] Based on the above results and the comparison of Ct values, it can be seen that the Ct values ​​of items 14-19 in Table 10 are significantly lower (i.e., earlier). Therefore, adding at least one nucleic acid stabilizer, either formamide or trehalose, along with either EDTA or EGTA, can not only improve the nucleic acid preservation performance of the entire system but also reduce the inhibitory effect of high-concentration surfactants on the entire reaction system. Considering the toxicity and availability of the components, trehalose and EDTA were chosen for use in subsequent experiments.

[0231] Example 5

[0232] pH plays a role in the release of pathogen nucleic acids and also affects the amplification of the reaction system. A stable pH ensures that the preservation solution functions effectively under various conditions. The basic formulation in this example is 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, and 1% (v / v) 0.5M EDTA (i.e., 5mM EDTA). The specific experimental setup is shown in Table 11.

[0233] Table 11

[0234] The specific operating procedure is as follows:

[0235] (1) Sample processing

[0236] Take a pseudovirus of influenza A virus and treat it with nuclease to ensure that the free nucleic acid inside is completely digested, leaving only intact virus particles.

[0237] (2) Sample release and nucleic acid extraction

[0238] The nuclease-digested pseudovirus was diluted 20-fold with the prepared preservation solution, and simultaneously diluted 20-fold with DEPC-treated water. The preservation solution containing the pseudovirus was thoroughly mixed and incubated at room temperature for 5 minutes. The pseudovirus treated with DEPC water was then extracted using a magnetic bead-based nucleic acid extraction and purification reagent.

[0239] (3) Sample testing

[0240] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0241] The results are shown in Table 12.

[0242] Table 12

[0243] The results showed that amplification was effective at pH values ​​between 9 and 12. However, when the pH was above 11, adding 1M Tris at 1.5% (v / v) affected the amplification results. The results indicated that the detection effect was best and highest at pH 11 with the addition of 1M Tris at 1.5% (v / v). Therefore, subsequent testing conditions were chosen with the addition of 0.5% (v / v) 1M Tris (i.e., 5mM Tris) and a pH adjustment to 11.0.

[0244] Example 6

[0245] The basic formulation in this embodiment is 0.1% (w / v) SDS, 0.2% (w / v) NP-40, 1% (w / v) trehalose, 1% (v / v) 0.5M EDTA (i.e., 5mM EDTA), and 0.5% (v / v) 1M Tris, with a solution pH of 11.0.

[0246] Based on the above results, the release capacity of samples containing common respiratory pathogens was validated. The samples included influenza A virus, adenovirus, mycoplasma pneumoniae, and human metapneumovirus.

[0247] The specific operating procedure is as follows:

[0248] (1) Sample processing

[0249] The samples were oropharyngeal swabs. Samples of influenza A virus, adenovirus, mycoplasma pneumoniae, and human metapneumovirus were mixed and prepared into a pooled sample.

[0250] (2) Sample release and nucleic acid extraction

[0251] The above samples were diluted 20-fold with preservation solution and DEPC-treated water, respectively. After thorough mixing, they were incubated at room temperature for 5 minutes. The samples diluted with DEPC-treated water were then extracted using a magnetic bead-based nucleic acid extraction and purification reagent.

[0252] (3) Sample testing

[0253] The above samples were tested using the Influenza A / Influenza B Virus Nucleic Acid Detection Kit (Fluorescent PCR Method), the Mycoplasma pneumoniae Nucleic Acid Detection Kit (Fluorescent PCR Method), and the Six Respiratory Virus Nucleic Acid Detection Kit (Fluorescent PCR Method), respectively.

[0254] The results are shown in Table 13.

[0255] Table 13

[0256] Based on the above results, the release efficiency of nucleic acids from DNA pathogens was better than that from RNA, possibly due to the higher pre-denaturation and denaturation temperatures in the amplification process, which facilitated the release of DNA nucleic acids. Based on these results, the existing preservation solution has a certain release efficiency for both DNA and RNA.

[0257] Example 7

[0258] Compared to relatively pure simulated samples, real clinical samples may contain more inhibitors. Therefore, this application adds grinding filter beads manufactured using a special process, the surface of which is modified with negatively charged hydroxyl or carboxyl groups. Simultaneously, the physical collision of the grinding filter beads improves the release efficiency of nucleic acids from the sample.

[0259] The preservation solution used in this embodiment consisted of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, and 0.5% (v / v) 1M Tris, with a pH of 11.0. 22 mL of the preservation solution was added to 11 sampling swabs and mixed thoroughly. The prepared preservation solution was dispensed into 2 mL tubes, and an equal amount of influenza A sample was added to each tube. Ten tubes were mixed thoroughly by vortexing with different masses of grinding beads, while one tube served as a control without grinding beads. 200 μL of this control was used as the sample for nucleic acid extraction. The amount of grinding beads added is shown in Table 14.

[0260] Table 14

[0261] The specific operating procedure is as follows:

[0262] (1) Sample processing

[0263] Take samples of influenza A virus

[0264] (2) Sample release and nucleic acid extraction

[0265] Take 22 mL of preservation solution, add 11 sampling swabs, and mix thoroughly. Aliquot the prepared preservation solution into 2 mL tubes, and add an equal amount of influenza A sample to each tube. Add different masses of grinding beads to 10 tubes and vortex thoroughly to mix. Use one tube without grinding beads as a control, and take 200 μL from the control tube as the sample for nucleic acid extraction.

[0266] (3) Sample testing

[0267] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0268] The results are shown in Table 15.

[0269] Table 15

[0270] Based on the above results, adding grinding filter beads of different qualities can improve the release efficiency of the sample.

[0271] Example 8

[0272] Based on the principle of grinding filter beads, other similar materials were selected for testing, including quartz sand, zirconia beads, foam filter beads, activated carbon particles, ceramsite, molecular sieves, and fiber balls. Their effects on impurity adsorption and sample release were evaluated after adding the preservation solution. The preservation solution used in this example consisted of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, and 0.5% (v / v) 1M Tris, with a pH of 11.0. The amount of grinding filter beads added is shown in Table 16.

[0273] Table 16

[0274] The specific operating procedure is as follows:

[0275] (1) Sample processing

[0276] Take samples of influenza A virus

[0277] (2) Sample release and nucleic acid extraction

[0278] Take 18 mL of preservation solution and add it to 9 sampling swabs, then mix thoroughly. Aliquot the prepared preservation solution into 2 mL tubes, and add an equal amount of influenza A sample to each tube. Process 8 tubes according to the table above, and use the remaining tube without the grinding filter beads as a control, from which 200 μL will be used as the sample for nucleic acid extraction.

[0279] (3) Sample testing

[0280] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0281] The results are shown in Table 17.

[0282] Table 17

[0283] Based on the above results, grinding filter beads of different materials can effectively improve the amplification effect after nucleic acid release from the sample. Therefore, using these materials in combination can be effective in different scenarios in practical applications.

[0284] Example 9

[0285] Even after adding grinding beads, the amplification effect of the preserved solution after sample release was still worse than that extracted by the magnetic bead method. Considering the significant impact of chemical reagents on the reaction system, physical methods were used to improve the pathogen detection effect. The preserved solution used in this example consisted of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, and 0.5% (v / v) 1M Tris, with a pH of 11.0. In the treatment group containing grinding beads, hydroxyl grinding beads were used, with a mass of 0.8g. The ultrasonic instrument parameters used in this study were 100W power and 35-40kHz frequency. The experimental protocol is shown in Table 18.

[0286] Table 18

[0287] The specific operating procedure is as follows:

[0288] (1) Sample processing

[0289] Take samples of influenza A virus

[0290] (2) Sample release and nucleic acid extraction

[0291] Take 10 mL of preservation solution, add it to 5 sampling swabs, and mix thoroughly. Aliquot the prepared preservation solution into 2 mL tubes, and add an equal amount of influenza A sample to each tube. Process 4 tubes according to the table above, and use the other tube as a control, from which 200 μL will be used as a sample for nucleic acid extraction.

[0292] (3) Sample testing

[0293] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0294] The results are shown in Table 19.

[0295] Table 19

[0296] Based on the above results, the amplification and detection effects of ultrasonic treatment combined with preservation solution and grinding beads are basically consistent with those of the magnetic bead method. However, if the preservation solution does not contain grinding beads after ultrasonic treatment, the amplification effect will actually be worse. This may be because the nucleic acid release efficiency is high after ultrasonication, but impurities are not removed, leading to the degradation of RNA and nucleic acid.

[0297] Example 10

[0298] Even after adding grinding beads, the amplification effect of the preserved solution after sample release was still worse than that extracted by the magnetic bead method. Considering the significant impact of chemical reagents on the reaction system, physical methods were used to assist in improving pathogen detection. The preserved solution used in this example consisted of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, and 0.5% (v / v) 1M Tris, with a pH of 11.0. In the treatment group containing grinding beads, hydroxyl grinding beads were used, with a mass of 0.8g. The ultrasonic instrument parameters used in this study were 100W power and 35-40kHz frequency. The experimental protocol is shown in Table 20.

[0299] Table 20

[0300] The specific operating procedure is as follows:

[0301] (1) Sample processing

[0302] Take samples of influenza A virus

[0303] (2) Sample release and nucleic acid extraction

[0304] Take 18 mL of preservation solution and add it to 9 sampling swabs, then mix thoroughly. Aliquot the prepared preservation solution into 2 mL tubes, and add an equal amount of influenza A sample to each tube. Process 8 tubes according to the table above, and use the remaining tube as a control, from which 200 μL will be used for nucleic acid extraction.

[0305] (3) Sample testing

[0306] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0307] The results are shown in Table 21.

[0308] Table 21

[0309] Based on the above results, different ultrasound times can improve the detection effect. Within the ultrasound range of 30-240 seconds, the release rate of the samples after ultrasounding reached more than 65%. However, excessively long ultrasound times may lead to a decrease in detection effect.

[0310] Example 11

[0311] Even after adding grinding beads, the amplification effect of the preserved solution after sample release was still worse than that extracted by the magnetic bead method. Considering the significant impact of chemical reagents on the reaction system, physical methods were used to improve the pathogen detection effect. The preserved solution used in this example consisted of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, and 0.5% (v / v) 1M Tris, with a pH of 11.0. In the treatment group containing grinding beads, hydroxyl grinding beads were used, with a mass of 0.8 g (2 ml). A metal bath was used for heating in this study. The experimental protocol is shown in Table 22.

[0312] Table 22

[0313] The specific operating procedure is as follows:

[0314] (1) Sample processing

[0315] Take samples of influenza A virus

[0316] (2) Sample release and nucleic acid extraction

[0317] Take 10 mL of preservation solution, add it to 5 sampling swabs, and mix thoroughly. Aliquot the prepared preservation solution into 2 mL tubes, and add an equal amount of influenza A sample to each tube. Process 4 tubes according to the table above, and use the other tube as a control, from which 200 μL will be used as a sample for nucleic acid extraction.

[0318] (3) Sample testing

[0319] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0320] The results are shown in Table 23.

[0321] Table 23

[0322] Based on the above results, the amplification and detection results of heat treatment combined with preservation solution and grinding beads are basically consistent with those of the magnetic bead method. However, if the preservation solution does not contain grinding beads after heat treatment, the amplification effect will actually be worse. This may be because the nucleic acid release efficiency is high after heating, but impurities are not removed, leading to the degradation of RNA and nucleic acids.

[0323] Example 12

[0324] Even after adding grinding beads, the amplification effect of the preserved solution after sample release was still worse than that extracted by the magnetic bead method. Considering the significant impact of chemical reagents on the reaction system, physical methods were used to improve pathogen detection. The preserved solution used in this example consisted of 0.1% SDS, 0.2% NP-40, 1% trehalose, 1% (v / v) 0.5M EDTA, and 0.5% (v / v) 1M Tris, with a pH of 11.0. In the treatment group containing grinding beads, hydroxyl grinding beads were used, with a mass of 0.8 g. A metal bath was used for heating in this study. The experimental protocol is shown in Table 24.

[0325] Table 24

[0326] The specific operating procedure is as follows:

[0327] (1) Sample processing

[0328] Take samples of influenza A virus

[0329] (2) Sample release and nucleic acid extraction

[0330] Take 20 mL of preservation solution, add 10 sampling swabs, and mix thoroughly. Aliquot the prepared preservation solution into 2 mL tubes, and add an equal amount of influenza A sample to each tube. Add different masses of grinding beads to 9 tubes and vortex thoroughly to mix. Use one tube without grinding beads as a control, and take 200 μL from it as the sample for nucleic acid extraction.

[0331] (3) Sample testing

[0332] The above samples were tested using an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method).

[0333] The results are shown in Table 25.

[0334] Table 25

[0335] Based on the above results, different heating times and temperatures can improve the detection effect. When heated to 75-95℃ for 1-3 minutes, the sample release rate can reach more than 80%.

[0336] Therefore, in practical applications, combining ultrasound or heating can further improve the nucleic acid release efficiency of this method and enhance the detection sensitivity of the entire detection system.

[0337] Comparative Example 1

[0338] Evaluate the reliability of this set of detection methods in hospital outpatient clinics, and compare the differences in detection results between using only the preservation solution, the preservation solution + grinding beads, and the preservation solution + grinding beads followed by ultrasonic treatment and magnetic bead nucleic acid extraction. The specific arrangements are as follows:

[0339] 1. After collecting samples using a sampling tube containing the preservation solution, directly perform detection, and at the same time take 200 μL for magnetic bead nucleic acid extraction.

[0340] 2. Add 0.8 g of hydroxy grinding beads to the samples in 1, let stand for 5 min, and then perform detection.

[0341] 3. After subjecting the samples in 2 to ultrasonic treatment, directly take samples for amplification.

[0342] Use an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method) to detect the above samples, and a total of 80 samples are compared. ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0354] Evaluate the reliability of this set of detection schemes in outpatient clinics of hospitals, and compare the differences in detection results between the preservation solution combined with ultrasonic treatment and the nucleic acid extraction by magnetic bead method. The specific arrangements are as follows:

[0355] 1. After collecting samples using a sampling tube containing a preservation solution, take 200 μL for nucleic acid extraction by magnetic bead method.

[0356] 2. After ultrasonic treatment of the samples placed in the preservation solution, take samples directly for amplification.

[0357] Use an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method) to detect the above samples, and a total of 30 samples are compared.

[0358] The specific operation process is as follows:

[0359] (1) Sample treatment and release

[0360] After collecting real oropharyngeal swab samples at the outpatient end and letting them stand for 5 minutes, take out 200 μL for nucleic acid extraction reagent by magnetic bead method. For the remaining samples, after processing with an ultrasonic device for 150 seconds, take out 5 μL for detection.

[0361] (2) Sample release and nucleic acid extraction

[0362] After taking out the above 200 μL samples, use nucleic acid extraction and purification reagents (Shanghai Feng Medical Equipment Preparation No. 20180202) for extraction.

[0363] (3) Sample detection

[0364] Use an influenza A virus / influenza B virus nucleic acid detection kit (fluorescent PCR method) to detect the above samples respectively.

[0365] The results are shown in Table 27.

[0366] Table 27

[0367] According to the above results, the total coincidence rate of using the preservation solution combined with ultrasonic treatment and the magnetic bead method can reach 90.00%. The detection results are better than those using only the preservation solution, and worse than those using the preservation solution combined with grinding beads and the preservation solution combined with grinding beads and additional ultrasonic treatment.

[0368] Discussion

[0369] In addition to having an excellent nucleic acid release rate, the method of this application has a short sample treatment time. Compared with the magnetic bead method, for the method of this application, after adding the sample to the preservation solution and letting it stand for 5 minutes, it can be detected using a nucleic acid detection kit, while the magnetic bead method involves steps such as magnetic bead adsorption, magnetic bead washing, and elution, which takes about 15 - 30 minutes.

[0370] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A formulation for sample preservation and nucleic acid release, comprising a preservation solution, said preservation solution being composed of a release component, a nucleic acid stabilizing component, and a buffer component; in, The releasing component consists of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component consists of 0.01%–10% nucleic acid protectant and 0.1–20 mM metal ion chelating agent; The buffer component is 0.1–50 mM tris(hydroxymethyl)aminomethane; The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof. The nonionic surfactant is selected from the group consisting of: nonylphenol polyoxyethylene ether, polysorbate-20, polyethylene glycol octylphenyl ether, polyoxyethylene lauroyl ether, or combinations thereof; The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof; The metal ion chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, or combinations thereof.

2. The formulation of claim 1, wherein, The preservation solution consists of 0.01%–5% sodium dodecyl sulfate, 0.01%–5% nonylphenol polyoxyethylene ether, 0.01%–10% trehalose, 0.1–10 mM ethylenediaminetetraacetic acid, and 0.1–50 mM tris(hydroxymethyl)aminomethane.

3. The formulation of claim 1, wherein, The preservation solution consists of 0.01%–3% sodium dodecyl sulfate, 0.02%–3% nonylphenol polyoxyethylene ether, 0.05%–5% trehalose, 3–8 mM ethylenediaminetetraacetic acid, and 1–30 mM tris(hydroxymethyl)aminomethane.

4. The formulation of claim 1, wherein, The formulation also includes ground filter beads.

5. The formulation of claim 4, wherein, The grinding filter beads are modified with hydroxyl or carboxyl groups.

6. The formulation of claim 4, wherein, The concentration of the ground filter beads in the formulation or the preservation solution is 0.01–5 g / mL.

7. The formulation of claim 4, wherein, The concentration of the ground filter beads in the formulation or the preservation solution is 0.05–3 g / mL.

8. The formulation of claim 4, wherein, The concentration of the ground filter beads in the formulation or the preservation solution is 0.1–2 g / mL.

9. The formulation of claim 4, wherein, The concentration of the ground filter beads in the formulation or the preservation solution is 0.2 to 1 g / mL.

10. A kit for sample preservation and nucleic acid release, comprising: A first container and a preservation solution located in the first container, the preservation solution being composed of a release component, a nucleic acid stabilizing component, and a buffer component; The releasing component consists of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant. The nucleic acid stabilizing component consists of 0.01%–10% nucleic acid protectant and 0.1–20 mM metal ion chelating agent; The buffer component is 0.1–50 mM tris(hydroxymethyl)aminomethane; The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof. The nonionic surfactant is selected from the group consisting of: nonylphenol polyoxyethylene ether, polysorbate-20, polyethylene glycol octylphenyl ether, polyoxyethylene lauroyl ether, or combinations thereof; The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof; The metal ion chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, or combinations thereof.

11. A method for increasing the nucleic acid release rate in a sample, comprising the following steps: (S1) Provide a copy; (S2) Add the sample to the preservation solution and mix well to preserve the sample and improve the nucleic acid release rate in the sample; The preservation solution consists of a release component, a nucleic acid stabilizing component, and a buffer component; in, The releasing component consists of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component consists of 0.01%–10% nucleic acid protectant and 0.1–20 mM metal ion chelating agent; The buffer component is 0.1–50 mM tris(hydroxymethyl)aminomethane; The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof. The nonionic surfactant is selected from the group consisting of: nonylphenol polyoxyethylene ether, polysorbate-20, polyethylene glycol octylphenyl ether, polyoxyethylene lauroyl ether, or combinations thereof; The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof; The metal ion chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, or combinations thereof.

12. The method of claim 11, wherein, The method further includes step (S3) adding grinding filter beads to the preservation solution containing the sample and mixing them.

13. A nucleic acid detection method, comprising the following steps: (z1) Add the sample to be tested into the preservation solution and mix well. The preservation solution consists of a release component, a nucleic acid stabilizing component and a buffer component. in, The releasing component consists of 0.01% to 5% anionic surfactant and 0.01% to 5% nonionic surfactant; The nucleic acid stabilizing component consists of 0.01%–10% nucleic acid protectant and 0.1–20 mM metal ion chelating agent; The buffer component is 0.1–50 mM tris(hydroxymethyl)aminomethane; The anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecylamine, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or combinations thereof. The nonionic surfactant is selected from the group consisting of: nonylphenol polyoxyethylene ether, polysorbate-20, polyethylene glycol octylphenyl ether, polyoxyethylene lauroyl ether, or combinations thereof; The nucleic acid protective agent is selected from the group consisting of formamide, trehalose, or combinations thereof; The metal ion chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, or combinations thereof; and (z2) Nucleic acid detection is performed on the preservation solution containing the sample to be tested to obtain the nucleic acid detection result.

Images