Viral nucleic acid sample diluent, viral nucleic acid sample extraction kit and viral nucleic acid extraction method

Abstract

This invention relates to the field of molecular detection technology, and in particular to viral nucleic acid sample diluents, viral nucleic acid sample extraction kits, and viral nucleic acid extraction methods. The viral nucleic acid sample release agent provided by this invention is compatible with various sampling kits. By adjusting the types and proportions of components, the critical micelle concentration of the surfactants used is minimized, ensuring lysis of cells and pathogens while reducing inhibitory effects on subsequent detection systems. With the addition of ion exchange resin, this invention can adsorb proteins and metal ions such as calcium and magnesium in the sample that affect subsequent reactions, promoting cell and viral lysis and enhancing system compatibility. The detection system of this invention has good compatibility, simultaneously compatible with multiple qPCR detection systems and also usable in isothermal RAA amplification systems. It is suitable not only for liquid detection systems but also for dry powder detection systems with large sample volumes.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to viral nucleic acid sample diluents, viral nucleic acid sample extraction kits, and viral nucleic acid extraction methods. Background Technology

[0002] Molecular detection technology has developed rapidly. Molecular diagnostics is a technology based on the detection of pathogen nucleic acids. It detects pathogens by recognizing their specific nucleic acid sequences. Therefore, obtaining suitable nucleic acids for the reaction becomes a crucial step in the accurate detection of pathogens, and the speed of nucleic acid extraction has become a bottleneck limiting the detection speed. Conventional nucleic acid extraction methods such as centrifuge column extraction and magnetic bead extraction not only require specialized instruments such as centrifuges and magnetic racks, but also involve relatively cumbersome steps, including lysis, washing, and elution.

[0003] With increasing testing pressure, the demand for home self-testing and rapid testing is gaining attention. This demand requires nucleic acid extraction procedures that are instrument-independent while possessing a certain level of detection capability. However, commercially available nucleic acid release agents do not perform as well as expected after releasing nucleic acid, and most are only designed for one detection method, failing to function properly in isothermal detection systems where rapid amplification is possible.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing nucleic acid release agents by providing a more flexible viral nucleic acid release agent with a wider range of applications. It is expected that this viral nucleic acid release agent can be used at room temperature and, when used with compatible consumables, can also be heated in an energy-free environment to achieve better nucleic acid release effects.

[0006] To solve the above-mentioned technical problems and achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a viral nucleic acid sample diluent, comprising: anionic surfactant, sodium hydroxide, EDTA, trehalose, and ion exchange resin, wherein the amount of EDTA added is 0-3% by mass-volume ratio, preferably 1%; the amount of the anionic surfactant added is 0.010%-0.050%, preferably 0.02%; and the amount of the ion exchange resin added is 0-5%, preferably 2.5%.

[0008] In an optional embodiment, the anionic surfactant is selected from sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or sodium octadecyl sulfate, preferably sodium hexadecyl sulfate.

[0009] In an optional embodiment, the concentration of sodium hydroxide is 0.16 to 1.28 mM, preferably 0.32 mM.

[0010] In optional embodiments, it also includes one or more combinations of polyols, sodium chloride, or NP-40.

[0011] In an optional embodiment, the polyol is selected from ethylene glycol or propylene glycol.

[0012] Preferably, the polyol is 0-10% propylene glycol by mass-volume ratio, and more preferably, the polyol is 5% propylene glycol.

[0013] Preferably, the concentration of NP-40 is 1% to 5% by mass-volume ratio, and more preferably 1%.

[0014] Preferably, the concentration of sodium chloride is 50-150 mM, and more preferably 100 mM.

[0015] In an optional embodiment, the ion exchange resin is selected from Chelex or Bio-Rex 70, wherein the Chelex has a specification of 50 to 400 mesh.

[0016] Preferably, the ion exchange resin is bio-rex 70.

[0017] In an optional embodiment, the trehalose concentration is 0.05–0.1 mM, preferably 0.075 mM.

[0018] Secondly, the present invention provides a viral nucleic acid sample extraction kit, comprising the viral nucleic acid sample diluent described in any of the foregoing embodiments.

[0019] Preferably, it also includes a heating device.

[0020] Thirdly, the present invention provides a method for extracting viral nucleic acid, wherein a viral sample is mixed with the viral nucleic acid sample diluent described in any of the foregoing embodiments, and viral nucleic acid is obtained after the reaction is completed.

[0021] Preferably, the reaction time is 5 to 15 minutes.

[0022] In an optional embodiment, the virus sample is mixed with the viral nucleic acid sample diluent described in any of the foregoing embodiments and then heated to obtain viral nucleic acid after the reaction is complete.

[0023] Preferably, the heating temperature is 90–110°C and the reaction time is 1–5 min.

[0024] The viral nucleic acid sample release agent provided by this invention is compatible with various sampling kits. Furthermore, by adjusting the types and proportions of components, the critical micelle concentration of the surfactants used is minimized, ensuring both cell and pathogen lysis while reducing inhibitory effects on subsequent detection systems. With the addition of ion exchange resin, this invention can adsorb proteins and metal ions such as calcium and magnesium that affect subsequent reactions, promoting cell and virus lysis and enhancing system compatibility.

[0025] In the process of providing viral nucleic acid samples, this invention can obtain viral nucleic acid by either placing the sample at room temperature for 5 minutes or by extracting it at 95°C for 3 minutes. The results of subsequent detection of the obtained nucleic acid are close to those of the extraction results of the magnetic bead method nucleic acid extraction reagent. At the same time, this extraction method can also be directly used to process swab samples and then perform nucleic acid amplification.

[0026] The detection system of this invention has good compatibility, being compatible with multiple qPCR detection systems simultaneously, and can also be used in isothermal RAA amplification systems. It is suitable not only for liquid detection systems but also for dry powder detection systems with large sample volumes.

[0027] When used in conjunction with self-heating consumables, this invention can provide a good nucleic acid release effect in a short time. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] In one specific embodiment, in a first aspect, the present invention provides a viral nucleic acid sample diluent, comprising: an anionic surfactant, sodium hydroxide, EDTA, trehalose, and an ion exchange resin. The amount of EDTA added, by mass-volume ratio, is 0-3%, including but not limited to 0.5%, 1%, 1.5%, 2%, 2.5%, or 3%, preferably 1%. The amount of the anionic surfactant added is 0.010%-0.050%, including but not limited to 0.010%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.050%, preferably 0.02%. The amount of the ion exchange resin added is 0-5%, including but not limited to 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, preferably 2.5%.

[0030] Most nucleic acid release agents on the market cannot be used in isothermal amplification or dry powder detection systems because of the lysis components they contain. This product solves this problem by reducing the concentration of the lysis components in adjacent micelles. It achieves good results in both RAA and qPCR detection methods and can be used in dry powder detection systems of the above detection technologies to further improve detection sensitivity.

[0031] EDTA in this invention is a common metal ion chelating agent, mainly used to reduce the inhibitors and metal ions that may exist in the sample and different sampling kits. Its physicochemical properties are stable, and it will not introduce other special metal ions except for Na ions. However, if the concentration of EDTA is too high, it will chelate magnesium ions in the subsequent amplification reaction, causing reaction inhibition.

[0032] The ion exchange resin in this invention mainly serves to assist in the lysis of cells and viruses and to adsorb impurities.

[0033] In an optional embodiment, the anionic surfactant is selected from sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate, or sodium octadecyl sulfate, preferably sodium hexadecyl sulfate.

[0034] The reason why sodium hexadecyl sulfate is preferred in this invention is that, according to the properties of surfactants, among surfactants of the same series with the same hydrophilic group, the larger the lipophilic group, the lower the critical micelle concentration. At the same time, from an economic point of view, sodium hexadecyl sulfate is preferred.

[0035] In an optional embodiment, the concentration of sodium hydroxide is 0.16 to 1.28 mM, including but not limited to 0.16 mM, 0.24 mM, 0.32 mM, 0.40 mM, 0.48 mM, 0.56 mM, 0.64 mM, 0.72 mM, 0.80 mM, 0.88 mM, 0.96 mM, 1.04 mM, 1.12 mM, 1.20 mM or 1.28 mM, preferably 0.32 mM.

[0036] In this invention, the alkaline environment is mainly provided by NaOH. In order to reduce the inhibition of the detection system, the concentration of NaOH is between 0.16 and 1.28 mM to provide an alkaline environment. The preferred concentration is 0.32 mM, which has the best effect.

[0037] In optional embodiments, it also includes one or more combinations of polyols, sodium chloride, or NP-40.

[0038] To further reduce the concentration of anionic surfactants and improve the compatibility of the system, this invention selects to add at least one of polyols, sodium chloride, or NP-40, and compound it with sodium hexadecyl sulfate to further reduce its critical micelle concentration.

[0039] In an optional embodiment, the polyol is selected from ethylene glycol or propylene glycol. These two polyols are highly polar and can competitively bind with water molecules, thus reducing the critical micelle concentration of the surfactant.

[0040] Preferably, the polyol is 0-10% propylene glycol by mass-volume ratio, including but not limited to 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%, and more preferably, the polyol is 5% propylene glycol.

[0041] Preferably, the concentration of NP-40 is 1% to 5% by mass-volume ratio, including but not limited to 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, preferably 1%;

[0042] Preferably, the concentration of sodium chloride is 50-150 mM, including but not limited to 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM or 150 mM, and preferably 100 mM.

[0043] In this invention, NP-40 and sodium chloride are both surfactant complexes, and these two substances have little adverse effect on the subsequent detection reaction.

[0044] In an optional embodiment, the ion exchange resin is selected from Chelex or Bio-Rex 70, preferably Bio-Rex 70.

[0045] The specifications of the Chelex range from 50 to 400 mesh, including but not limited to 50 mesh, 55 mesh, 60 mesh, 65 mesh, 70 mesh, 75 mesh, 80 mesh, 85 mesh, 90 mesh, 95 mesh, 100 mesh, 110 mesh, 120 mesh, 130 mesh, 140 mesh, 150 mesh, 160 mesh, 170 mesh, 180 mesh, 190 mesh, 200 mesh, 220 mesh, 240 mesh, 260 mesh, 280 mesh, 300 mesh, 330 mesh, 360 mesh, 390 mesh, or 400 mesh.

[0046] In an optional embodiment, the trehalose concentration is 0.05 to 0.1 mM, including but not limited to 0.05 mM, 0.055 mM, 0.06 mM, 0.065 mM, 0.07 mM or 0.075 mM, preferably 0.075 mM.

[0047] Trehalose, a commonly used PCR enhancer, is added to the system primarily to enhance its stability.

[0048] In a second aspect, the present invention provides a viral nucleic acid sample extraction kit, comprising the viral nucleic acid sample diluent described in any of the foregoing embodiments;

[0049] Preferably, it also includes a heating device.

[0050] Thirdly, the present invention provides a method for extracting viral nucleic acid, wherein a viral sample is mixed with the viral nucleic acid sample diluent described in any of the foregoing embodiments, and viral nucleic acid is obtained after the reaction is completed.

[0051] Preferably, the reaction time is 5 to 15 minutes, including but not limited to 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes.

[0052] In an optional embodiment, the virus sample is mixed with the viral nucleic acid sample diluent described in any of the foregoing embodiments and then heated to obtain viral nucleic acid after the reaction is complete.

[0053] Preferably, the heating temperature is 90 to 110°C, including but not limited to 90°C, 92°C, 94°C, 96°C, 98°C, 100°C, 102°C, 104°C, 106°C, 108°C or 110°C, and the reaction time is 1 to 5 min, including but not limited to 1 min, 2 min, 3 min, 4 min or 5 min.

[0054] For example, when using at room temperature: mix the virus sample and the virus nucleic acid sample release agent at a volume ratio of 1:1, mix thoroughly, let stand for 10 minutes, and then take the mixture for subsequent testing.

[0055] For example, when using under heating conditions: mix the virus sample and the viral nucleic acid release agent at a volume ratio of 1:1, mix thoroughly, place in a metal bath and heat at 95°C for 3 minutes, then take the mixture for subsequent testing.

[0056] Alternatively, use with the accompanying heating pack: Mix the virus sample and viral nucleic acid release agent in a 1:1 volume ratio in a 1.5ml centrifuge tube, mix thoroughly, add about 5ml of tap water or mineral water to the heating pack, place the 1.5ml centrifuge tube in the heating pack and heat for 3 minutes. After the time is up, wait for the 1.5ml EP tube to cool slightly, and then take the mixture for subsequent experiments.

[0057] The following describes some embodiments of the present invention in detail. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0058] Example 1

[0059] This embodiment provides a viral nucleic acid sample release agent, composed of the following components: NaOH 0.16–1.28 mM, sodium dodecyl sulfate 0.5% (w / v), propylene glycol 10% (w / v), NP-40 5% (w / v), NaCl 150 mM, EDTA 0%–3% (w / v), trehalose 0.05–0.1 mM, and bio-rex70 5% (w / v). The specific testing protocol is as follows:

[0060]

[0061]

[0062] Experimental methods:

[0063] (1) Sample preparation

[0064] Take a fake COVID-19 virus, with an initial concentration of 10. 6 The concentration is 100 mg / ml, diluted with the Youkang Hengye sampling kit used after a negative swab collection, to a final concentration of 100 mg / ml. 5 cope / ml is available for later use.

[0065] (2) Sample processing and detection

[0066] The prepared spurious virus samples were subjected to room temperature and heat treatment respectively using different formulations of viral nucleic acid sample release agents as described in this embodiment. The sample volume was 20 μL, and the release dose was 20 μL. For room temperature treatment, the spurious virus sample and viral nucleic acid sample release agent were mixed thoroughly and allowed to stand at room temperature for 10 min. For heat treatment, the spurious virus sample and viral nucleic acid sample release agent were mixed thoroughly and placed in a 95℃ metal bath for 3 min. All extracted nucleic acids were detected using a commercially available novel coronavirus nucleic acid detection kit (fluorescent PCR method) (Shanghai Berger Medical Technology Co., Ltd.).

[0067] The test results are as follows:

[0068]

[0069]

[0070]

[0071]

[0072] The above test results show that the preferred concentration of NaOH is 0.32 mM, the preferred concentration of EDTA is 1%, and the preferred concentration of trehalose is 0.075 mM.

[0073] It should be noted that the role of EDTA in the system depends on the sampling tube used. If the sampling tube contains water or has a simple composition, it may not need to be added. However, for most non-inactivated sampling tubes, it needs to be added. Based on the above experimental results, 1% of the diluent provided in this invention is sufficient to chelate the metal ions. Adding more will inhibit the reaction.

[0074] Example 2

[0075] This embodiment provides a viral nucleic acid sample release agent, composed of the following components: NaOH 0.32mM, anionic surfactants (including but not limited to sodium dodecyl sulfate, sodium tetradecyl sulfate, and sodium hexadecyl sulfate) 0.01-0.05% (w / v), propylene glycol 10% (w / v), NP-40 5% (w / v), NaCl 150mM, EDTA 1% (w / v), trehalose 0.075mM, and bio-rex 70 5% (w / v). The specific testing protocol is as follows:

[0076] Types of surfactants concentration Formula 1 Sodium dodecyl sulfate 0.050％ Formula 2 Sodium tetradecyl sulfate 0.050％ Formula 3 Sodium hexadecyl sulfate 0.050％ Formula 4 Sodium dodecyl sulfate 0.1％ Formula 5 Sodium tetradecyl sulfate 0.1％ Formula Six Sodium hexadecyl sulfate 0.1％

[0077] The experimental method is the same as in Example 1, and the detection results are as follows:

[0078]

[0079]

[0080] Heat treatment Types of surfactants concentration Repeat 1 Repeat 2 average value Formula 1 Sodium dodecyl sulfate 0.050％ O gene 31.06 31.08 31.07 N gene 32.09 32.09 32.09 Formula 2 Sodium tetradecyl sulfate 0.050％ O gene 31.08 31.10 31.09 N gene 32.07 32.09 32.08 Formula 3 Sodium hexadecyl sulfate 0.050％ O gene 31.05 31.08 31.07 N gene 32.09 32.11 32.10 Formula 4 Sodium dodecyl sulfate 0.1％ O gene 34.03 34.50 34.27 N gene 35.53 35.71 35.62 Formula 5 Sodium tetradecyl sulfate 0.1％ O gene 33.99 34.22 34.11 N gene 34.86 34.76 34.81 Formula Six Sodium hexadecyl sulfate 0.1％ O gene 34.02 34.32 34.17 N gene 35.34 34.68 35.01

[0081] The above test results show that sodium hexadecyl sulfate is the preferred anionic surfactant. Under the condition that the release and detection performance are the same, the longer the hydrocarbon chain, the lower the critical micelle concentration.

[0082] Example 3

[0083] This embodiment provides a viral nucleic acid sample release agent, composed of the following components: NaOH 0.32mM, sodium hexadecyl sulfate 0.01-0.05% (w / v), propylene glycol 0-10% (w / v), NP-40 1-5% (w / v), NaCl 50-150mM, EDTA 1% (w / v), trehalose 0.075mM, and bio-rex70 5% (w / v). The specific testing protocol is as follows:

[0084] Sodium hexadecyl sulfate Propylene glycol Sodium chloride NP-40 Formula 1 0.010％ 0％ 50mM 1％ Formula 2 0.010％ 5％ 150mM 3％ Formula 3 0.010％ 10％ 100mM 5％ Formula 4 0.025％ 0％ 150mM 5％ Formula 5 0.025％ 5％ 100mM 1％ Formula Six 0.025％ 10％ 50mM 3％ Formula Seven 0.050％ 0％ 100mM 3％ Formula 8 0.050％ 5％ 50mM 5％ Formula Nine 0.050％ 10％ 150mM 1％

[0085] The experimental method is the same as in Example 1, and the detection results are as follows:

[0086]

[0087]

[0088] heating Sodium hexadecyl sulfate Propylene glycol Sodium chloride NP-40 target Repeat 1 Repeat 2 average value Formula 1 0.010％ 0％ 50mM 1％ O gene 30.05 30.21 30.13 N gene 31.19 31.17 31.18 Formula 2 0.010％ 5％ 150mM 3％ O gene 30.09 30.13 30.11 N gene 31.24 31.21 31.23 Formula 3 0.010％ 10％ 100mM 5％ O gene 32.22 31.54 31.88 N gene 32.95 33.45 33.20 Formula 4 0.025％ 0％ 150mM 5％ O gene 29.93 30.07 30 N gene 30.84 30.73 30.78 Formula 5 0.025％ 5％ 100mM 1％ O gene 29.08 29.06 29.07 N gene 30.13 30.14 30.14 Formula Six 0.025％ 10％ 50mM 3％ O gene 31.37 31.21 31.29 N gene 32.50 31.93 32.21 Formula Seven 0.05％ 0％ 100mM 3％ O gene 30.13 30.2 30.16 N gene 31.05 31.05 31.05 Formula 8 0.05％ 5％ 50mM 5％ O gene 30.13 30.23 30.18 N gene 31.08 31.03 31.05 Formula Nine 0.05％ 10％ 150mM 1％ O gene 32.36 31.54 31.95 N gene 33.14 32.62 32.88

[0089] The above test results show that the preferred concentrations are: sodium hexadecyl sulfate 0.025%, propylene glycol 5%, NaCl 100mM, and NP-40 1%.

[0090] Example 4

[0091] This embodiment provides a viral nucleic acid sample release agent, composed of the following components: NaOH 0.32mM, sodium hexadecyl sulfate 0.025% (w / v), propylene glycol 5% (w / v), NP-40 1% (w / v), NaCl 100mM, EDTA 1% (w / v), trehalose 0.075mM, and ion exchange resin 0-5% (w / v), including Chelex-100 (50-100 mesh), Chelex-100 (100-200 mesh), Chelex-100 (200-400 mesh), and Bio-Rex 70. The specific testing protocol is as follows:

[0092] Types of cation exchange resins concentration Formula 1 bio-rex 0％ Formula 2 bio-rex 2.50％ Formula 3 bio-rex 5％ Formula 4 Chelex 100 (50-100 mesh) 2.50％ Formula 5 Chelex 100 (100-200 mesh) 2.50％ Formula Six Chelex 100 (200-400 mesh) 2.50％

[0093] The experimental method is the same as in Example 1, and the detection results are as follows:

[0094] normal temperature Types of ion exchange resins concentration target Repeat 1 Repeat 2 average value Formula 1 bio-rex 0％ O gene 32.46 32.45 32.45 N gene 33.57 33.53 33.55 Formula 2 bio-rex 2.50％ O gene 30.54 30.41 30.47 N gene 31.76 31.61 31.69 Formula 3 bio-rex 5％ O gene 30.46 30.39 30.42 N gene 31.70 31.61 31.65 Formula 4 Chelex 100 (50-100 mesh) 2.50％ O gene 30.54 30.34 30.44 N gene 31.54 31.69 31.61 Formula 5 Chelex 100 (100-200 mesh) 2.50％ O gene 30.41 30.41 30.41 N gene 31.76 31.57 31.66 Formula Six Chelex 100 (200-400 mesh) 2.50％ O gene 30.32 30.35 30.33 N gene 31.79 31.55 31.67

[0095]

[0096]

[0097] Repeat the above sample processing and nucleic acid extraction experiments, and use the Novel Coronavirus Nucleic Acid Detection Kit (Isothermal Amplification Method) (Shanghai Berger Medical Technology Co., Ltd.) on an isothermal nucleic acid amplification and detection analyzer (BG-Nova-X8) to determine positive and negative results. The test results are as follows:

[0098] normal temperature Types of cation exchange resins concentration Repeat 1 Repeat 2 Formula 1 bio-rex 0％ Positive Positive Formula 2 bio-rex 2.50％ Positive Positive Formula 3 bio-rex 5％ Positive Positive Formula 4 Chelex 100 (50-100 mesh) 2.50％ Positive Positive Formula 5 Chelex 100 (100-200 mesh) 2.50％ Positive Positive Formula Six Chelex 100 (200-400 mesh) 2.50％ Positive Positive

[0099]

[0100]

[0101] The test results above show that the preferred ion exchange resin is BIRR-REX70, and the preferred concentration is 2.5%.

[0102] Example 5

[0103] This embodiment provides a viral nucleic acid sample release agent with the following composition:

[0104] Components concentration NaOH 3.2mM Sodium hexadecyl sulfate 0.025％ Propylene glycol 5％ NP-40 1％ NaCl 100mM 0.5M EDTA 1% (v / v) Bio-rex 70 2.5% (w / v) Trehalose 0.075mM

[0105] Using the viral nucleic acid sample release agent provided in this embodiment, the pseudovirus sample was processed according to the experimental method provided in Example 1, and the extracted sample was detected by qPCR detection reagent and RAA detection reagent respectively.

[0106] Comparative Example 1

[0107] The viral nucleic acid release agent provided in this comparative example includes 25mM NaOH, 1.0% Triton X 100, 2mM EDTA and 10mM Tris HCl. The method of use is as follows: take 20μL of pseudovirus sample, add 20μL of viral nucleic acid release agent, vortex mix, let stand at room temperature for 1 min, and then use the mixture.

[0108] Comparative Example 2

[0109] Purchase Virus Nucleic Acid Sample Release Agent Product 1 (Catalog No.: WLDR8202-S) from Weifang Anpu Future Biotechnology Co., Ltd. The usage method is as follows: Take 20 μL of pseudovirus sample, add 5 μL of Virus Nucleic Acid Sample Release Agent Product 1, and mix gently; place the mixture in a metal bath and incubate at 95℃ for 5 min; remove the sample and equilibrate at room temperature for 3 min, then centrifuge at 10000 rpm for 2 min; use the supernatant directly for subsequent reactions.

[0110] Comparative Example 3

[0111] DEPC water was used as the viral nucleic acid sample release agent. 20 μL of pseudovirus sample was taken, 20 μL of viral nucleic acid sample release agent was added, vortexed and mixed, and then left at room temperature for 5 min before subsequent detection.

[0112] Comparative Example 4

[0113] In this comparative example, the viral nucleic acid sample release agent consisted of 50 mmol / L guanidine isothiocyanate, 0.05% Tween-20 (v / v), 0.05% Triton X-100, 25% ethanol, 20% isoamyl alcohol, and enzyme-free sterile water. The method of use was to mix the viral nucleic acid sample release agent with the viral sample at a 1:1 ratio, incubate for 30 minutes, and then use it for subsequent detection.

[0114] Comparative Example 5

[0115] Purchased from Guangzhou Meiji Biotechnology Co., Ltd. Magnetic bead nucleic acid extraction reagent, Guangdong Sui Medical Device Registration No. 20150062.

[0116] Comparative Example 6

[0117] In this comparative example, the viral nucleic acid sample release agent included a molar concentration of NaOH of 0.1 M, a volume percentage (mL / mL) of NP40 of 1%, a mass volume percentage (mg / mL) of LLS of 0.5%, a molar concentration of guanidine isothiocyanate of 0.1 M, a mass volume percentage of SDS of 0.2%, and a mass volume percentage of Foam ban of 0.5%.

[0118] The extraction effects of Examples 5 and Comparative Examples 1-6 were tested using the following experimental methods:

[0119] (1) Sample preparation:

[0120] Take a fake COVID-19 virus, with an initial concentration of 10. 6 The concentration is 100 mg / ml, diluted with the Youkang Hengye sampling kit used after a negative swab collection, to a final concentration of 100 mg / ml. 5 cope / ml is available for later use.

[0121] (2) Sample processing and nucleic acid extraction:

[0122] The prepared fake virus samples were subjected to room temperature and heat treatment respectively using the viral nucleic acid sample release agent described in Example 5 of this invention. Simultaneously, the samples were treated with the viral nucleic acid sample release agents provided in Comparative Examples 1, 2, 3, 4, and 6 respectively. 200 μL of sample from Comparative Example 5 was extracted. The extracted nucleic acids were all detected using commercially available novel coronavirus nucleic acid detection kits (fluorescent PCR method) (Shanghai Berger Medical Technology Co., Ltd.) and (Guangzhou Da An Gene Co., Ltd.).

[0123] Repeat the above sample processing and nucleic acid extraction experiments, and use the novel coronavirus nucleic acid detection kit (isothermal amplification method) (Shanghai Berger Medical Technology Co., Ltd.) on the isothermal nucleic acid amplification detection analyzer (BG-Nova-X8) to determine the positive or negative result.

[0124] The results of the novel coronavirus nucleic acid detection kit (fluorescent PCR method) (Shanghai Berger Medical Technology Co., Ltd.) are as follows:

[0125]

[0126]

[0127] The results of the novel coronavirus nucleic acid detection kit (fluorescent PCR method) (Guangzhou Da An Gene Co., Ltd.) are as follows:

[0128] target Repeat 1 Repeat 2 average value Example 5 - Room Temperature O gene 20.41 20.94 20.67 N gene 21.09 21.51 21.30 Example 5 - Heating O gene 19.01 19.01 19.01 N gene 19.55 19.59 19.57 Comparative Example 1 O gene 23.75 24.37 24.06 N gene 24.13 24.45 24.29 Comparative Example 2 O gene 23.57 24.68 24.12 N gene 23.75 24.88 24.32 Comparative Example 3 O gene 23.81 24.29 24.05 N gene 24.26 24.39 24.32 Comparative Example 4 O gene 27.57 25.15 26.36 N gene 27.87 25.18 26.52 Comparative Example 5 O gene 21.34 21.63 21.48 N gene 21.68 21.87 21.78 Comparative Example 6 O gene 22.32 22.51 21.48 N gene 22.89 22.96 22.93

[0129] The results of the novel coronavirus nucleic acid detection kit (isothermal amplification method) (Shanghai Berger Medical Technology Co., Ltd.) are as follows:

[0130] Repeat 1 Repeat 2 Example 5 - Room Temperature O gene Positive Positive N gene Positive Positive Example 5 - Heating O gene Positive Positive N gene Positive Positive Comparative Example 1 O gene Negative Negative N gene Negative Negative Comparative Example 2 O gene Negative Negative N gene Negative Negative Comparative Example 3 O gene Negative Negative N gene Negative Negative Comparative Example 4 O gene Positive Negative N gene Positive Positive Comparative Example 5 O gene Positive Positive N gene Positive Positive Comparative Example 5 O gene Negative Negative N gene Negative Negative Comparative Example 6 O gene Negative Negative N gene Negative Negative

[0131] The experimental results above show that the viral nucleic acid sample release agent provided by this invention can achieve good detection results in qPCR detection reagents from different manufacturers. It also yields good experimental results in isothermal RAA detection.

[0132] Example 6

[0133] Three commercially available non-inactivated sampling kits were selected. The viral nucleic acid sample release agent provided in Example 5 was used to treat diluted pseudovirus samples from the three sampling kits under both room temperature and heat treatment conditions. The samples were then detected using qPCR and RAA detection reagents, respectively. The experimental methods are as follows:

[0134] (1) Sample preparation:

[0135] Take a fake COVID-19 virus, with an initial concentration of 10. 6 The concentration was diluted to 10^3 / ml using three non-inactivated sampling kits obtained from negative swab collection. 5 cope / ml is available for later use.

[0136] (2) Sample processing and nucleic acid extraction:

[0137] The prepared spurious virus samples were treated at room temperature and with heat using the sample release agent described in this invention. The extracted nucleic acids were analyzed using a commercially available novel coronavirus nucleic acid detection kit (fluorescent PCR method) (Shanghai Berger Medical Technology Co., Ltd.).

[0138] Repeat the above sample processing and nucleic acid extraction experiments, and use the novel coronavirus nucleic acid detection kit (isothermal amplification method) (Shanghai Berger Medical Technology Co., Ltd.) on the isothermal nucleic acid amplification detection analyzer (BG-Nova-X8) to determine the positive or negative result.

[0139] The results of the novel coronavirus nucleic acid detection kit (fluorescent PCR method) (Shanghai Berger Medical Technology Co., Ltd.) are as follows:

[0140]

[0141]

[0142] heating target Repeat 1 Repeat 2 AVG Manufacturer 1 O gene 29.11 29.4 29.26 N gene 30.05 30.14 30.10 Manufacturer 2 O gene 29.55 29.76 29.66 N gene 31.06 30.98 31.02 Manufacturer 3 O gene 29.04 29.57 29.31 N gene 30.98 30.67 30.83

[0143] The results of the novel coronavirus nucleic acid detection kit (isothermal amplification method) (Shanghai Berger Medical Technology Co., Ltd.) are as follows:

[0144] Repeat 1 Repeat 2 Manufacturer 1 Positive Positive Manufacturer 2 Positive Positive Manufacturer 3 Positive Positive

[0145] The experimental results above show that the viral nucleic acid sample release agent provided by this invention can be adapted to qPCR and RAA detection in sampling kits from three different manufacturers.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A viral nucleic acid sample diluent, characterized in that, It is composed of the following components: 0.010%~0.050% anionic surfactant, 0.16~1.28mM sodium hydroxide, 0~3% EDTA, 0.05~0.1mM trehalose, 2.5~5% ion exchange resin, 1%~5% NP-40, 0~10% polyol and 50~150mM sodium chloride; The concentrations of anionic surfactants, EDTA, ion exchange resins, NP-40, and polyols are expressed as mass-volume ratios. The anionic surfactant is sodium hexadecyl sulfate; The polyol is propylene glycol; The ion exchange resin is bio-rex 70.

2. The viral nucleic acid sample diluent according to claim 1, characterized in that, The concentration of sodium hydroxide is 0.32 mM.

3. The viral nucleic acid sample diluent according to claim 1, characterized in that, According to the mass-volume ratio, the polyol is 5% propylene glycol.

4. The viral nucleic acid sample diluent according to claim 1, characterized in that, The concentration of NP-40 is 1% by mass-volume ratio.

5. The viral nucleic acid sample diluent according to claim 1, characterized in that, The concentration of sodium chloride is 100 mM.

6. The viral nucleic acid sample diluent according to claim 1, characterized in that, The trehalose concentration was 0.075 mM.

7. The viral nucleic acid sample diluent according to claim 1, characterized in that, The amount of EDTA added is 1%.

8. The viral nucleic acid sample diluent according to claim 1, characterized in that, The amount of the anionic surfactant added is 0.02%.

9. The viral nucleic acid sample diluent according to claim 1, characterized in that, The amount of ion exchange resin added is 2.5%.

10. A viral nucleic acid sample extraction kit, characterized in that, Includes the viral nucleic acid sample diluent according to any one of claims 1 to 9.

11. The viral nucleic acid sample extraction kit according to claim 10, characterized in that, It also includes a heating device.

12. A method for extracting viral nucleic acid, characterized in that, The virus sample is mixed with the viral nucleic acid sample diluent according to any one of claims 1 to 9, and the viral nucleic acid is obtained after the reaction is completed.

13. The viral nucleic acid extraction method according to claim 12, characterized in that, The reaction time is 5-15 minutes.

14. The extraction method according to claim 13, characterized in that, The virus sample is mixed with the viral nucleic acid sample diluent according to any one of claims 1 to 9 and heated. After the reaction is completed, viral nucleic acid is obtained.

15. The extraction method according to claim 14, characterized in that, The heating temperature is 90~110℃, and the reaction time is 1~5min.

Images