Nucleic acid releasing agent, preparation method and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a nucleic acid releasing agent and a preparation method and application thereof. The invention provides a nucleic acid releasing agent. The nucleic acid releasing agent comprises chaotropic salt, polyethylene glycol, a surfactant, an antifreeze agent, a penetrant, a metal chelating agent, an antioxidant and a buffer solution. According to the invention, the extraction-free rapid nucleic acid releasing agent is researched and developed, and an ultrasonic treatment scheme is introduced to carry out rapid pretreatment on the sample, so that the problems of tedious nucleic acid extraction process, long time consumption and low efficiency in the prior art are successfully solved, and an efficient, convenient and low-cost nucleic acid releasing and detecting scheme is provided for clinical detection and scientific research.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to nucleic acid releasing agents, preparation methods, and applications. Background Technology

[0002] Nucleic acids, including DNA and RNA, are essential components of biological molecules, and nucleic acid testing is a crucial step in pathogen detection and screening. Especially in today's precision medicine and prevention strategies for respiratory diseases, nucleic acid testing plays a vital role as the "gold standard." However, the nucleic acid extraction step in traditional nucleic acid testing procedures often becomes a bottleneck restricting testing efficiency. This process is not only cumbersome and complex, involving multiple intricate steps, but also time-consuming and requires strict control over experimental conditions and operator skills, increasing testing costs and the risk of human error. Furthermore, the inefficiency of traditional nucleic acid extraction methods is particularly pronounced when facing the large-scale screening needs of sudden outbreaks, making it difficult to meet the requirements for rapid and accurate testing.

[0003] Therefore, the industry urgently needs a new technology that can bypass cumbersome extraction steps and directly achieve efficient nucleic acid release, simplifying the testing process, shortening testing time, and reducing the professional requirements for operators, thereby promoting the innovation and popularization of nucleic acid testing technology. The extraction-free rapid nucleic acid release agent of this invention was developed based on this urgent need, aiming to provide powerful tool support for rapid disease diagnosis through technological innovation. Summary of the Invention

[0004] In view of this, the present invention provides a nucleic acid release agent, a preparation method, and its applications. By developing an extraction-free rapid nucleic acid release agent and introducing an ultrasonic treatment scheme for rapid sample pretreatment, the present invention successfully solves the problems of cumbersome, time-consuming, and inefficient nucleic acid extraction processes in existing technologies, providing an efficient, convenient, and low-cost nucleic acid release and detection solution for clinical testing and scientific research.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a nucleic acid release agent comprising the following components:

[0007]

[0009] In some embodiments of the present invention, the nucleic acid releasing agent described above includes one or more of guanidine thiocyanate, guanidine hydrochloride, and potassium thiocyanate.

[0010] In some embodiments of the present invention, the ionizing salt in the above-mentioned nucleic acid releasing agent is guanidine thiocyanate.

[0011] In some embodiments of the present invention, the surfactant in the above-mentioned nucleic acid releasing agent includes one or more of Triton X-100, sodium dodecyl sulfonate, and CHAPS.

[0012] In some embodiments of the present invention, the surfactant in the above-mentioned nucleic acid releasing agent is Triton X-100.

[0013] In some embodiments of the present invention, the antifreeze agent in the above-mentioned nucleic acid releasing agent includes one or more of ethylene glycol, propylene glycol and glycerol.

[0014] In some embodiments of the present invention, the antifreeze agent in the above-mentioned nucleic acid releasing agent is glycerol.

[0015] In some embodiments of the present invention, the permeabilizer in the above-mentioned nucleic acid releasing agent includes one or more of methyl sulfoxide, dimethyl sulfoxide, and decyl methyl sulfoxide.

[0016] In some embodiments of the present invention, the permeabilizer in the above-mentioned nucleic acid releasing agent is dimethyl sulfoxide.

[0017] In some embodiments of the present invention, the metal chelating agent in the above-mentioned nucleic acid releasing agent includes one or more of EGTA, EDTA and DTPA.

[0018] In some embodiments of the present invention, the metal chelating agent in the above-mentioned nucleic acid releasing agent is EGTA.

[0019] In some embodiments of the present invention, the antioxidant in the above-mentioned nucleic acid releasing agent includes one or more of vitamin C, glutathione, and tea polyphenols.

[0020] In some embodiments of the present invention, the antioxidant in the above-mentioned nucleic acid releasing agent is glutathione.

[0021] In some embodiments of the present invention, the buffer solution in the above-mentioned nucleic acid release agent includes: Tris-HCl buffer and / or sodium citrate buffer.

[0022] In some embodiments of the present invention, the buffer solution in the above-mentioned nucleic acid releasing agent is Tris-HCl buffer solution.

[0023] In some embodiments of the present invention, the polyethylene glycol in the above-mentioned nucleic acid releasing agent is: polyethylene glycol-n, n=200~20000, preferably n=2000.

[0024] In some embodiments of the present invention, the above-mentioned nucleic acid releasing agent comprises the following components:

[0025]

[0027] In some embodiments of the present invention, the above-mentioned nucleic acid releasing agent comprises the following components:

[0028]

[0029]

[0031] The present invention also provides a method for preparing the above-mentioned nucleic acid release agent, comprising the following steps: mixing the components and adjusting the pH value to obtain the nucleic acid release agent.

[0032] In some embodiments of the present invention, in the above preparation method, the pH value is adjusted to 6-9.

[0033] In some embodiments of the present invention, the above preparation method includes the following steps: dissolving guanidine thiocyanate and mixing it with Tris-HCl buffer, then sequentially adding PEG2000, EGTA, glutathione, Triton-100, DMSO and glycerol, adjusting the pH value, and then bringing the volume to a final volume to obtain the nucleic acid release agent.

[0034] The present invention also provides the application of the above-described nucleic acid releasing agent and / or the nucleic acid releasing agent obtained by the above preparation method in any of the following:

[0035] (a) Nucleic acid extraction; and / or

[0036] (b) Preparation of nucleic acid extraction products; and / or

[0037] (c) Nucleic acid testing; and / or

[0038] (d) Preparation of nucleic acid testing products.

[0039] In some embodiments of the present invention, the above-described application includes the following steps: mixing the sample to be tested with the nucleic acid releasing agent, sonicating, and obtaining the sample.

[0040] In some embodiments of the present invention, in the above applications, the frequency of the ultrasound is 10~100KHz and the duration is 1~60s.

[0041] In some embodiments of the present invention, in the above applications, the frequency of the ultrasound is 40~50KHz and the duration is 10~30s.

[0042] The present invention also provides detection reagents and / or detection kits, comprising: the above-described nucleic acid releasing agent and / or the nucleic acid releasing agent obtained by the above preparation method, as well as acceptable adjuvants, carriers and / or devices.

[0043] The present invention also provides a method for using the above-mentioned detection reagents and / or detection kits, wherein the detection reagents and / or detection kits are mixed with the sample to be tested and then sonicated.

[0044] In some embodiments of the present invention, in the above-described method of use, the frequency of the ultrasound is 10~100KHz and the duration is 1~60s.

[0045] In some embodiments of the present invention, in the above-described method of use, the frequency of the ultrasound is 40~50KHz and the duration is 10~30s.

[0046] The beneficial effects of this invention include:

[0047] (1) Simple operation: No extraction required. It eliminates the complicated extraction steps in traditional nucleic acid testing, eliminates the need for professional technicians to perform tedious operations, greatly simplifies the nucleic acid testing process, saves time and labor costs, improves testing efficiency, and is suitable for large-scale testing applications.

[0048] (2) High efficiency and convenience: Through the combined action of ultrasonic treatment and extraction-free rapid nucleic acid release agent, rapid release and efficient detection of nucleic acid are achieved. The nucleic acid release process can be completed in about 30 seconds, which greatly shortens the detection cycle compared with traditional methods. It can provide timely basis for clinical diagnosis, and its advantages are particularly obvious in emergency situations such as disease prevention and control.

[0049] (3) Sufficient release: It can effectively release nucleic acid in the sample, ensuring the accuracy and reliability of nucleic acid detection. Through testing on different types of samples, it can achieve efficient nucleic acid release, providing sufficient and high-quality nucleic acid templates for subsequent testing.

[0050] (4) Wide applicability: This technical solution is applicable to a variety of sample types, including difficult-to-process samples such as sputum and whole blood, providing more possibilities for clinical testing and scientific research.

[0051] (5) Special components prevent nucleic acid degradation: The nucleic acid release agent of this invention innovatively introduces a multi-component protection system. Through the combined action of chelating agents, reducing agents, and nucleic acid stabilizers, the stability of nucleic acids after release from different samples is greatly improved. By adding specific components, divalent metal ions such as Mg²⁺ and Ca²⁺ in the sample can be rapidly chelated, inhibiting the activity of DNase and RNase, preventing oxidative damage, and maintaining the stability of the nucleic acid double helix structure through osmotic regulation. In particular, it forms a protective barrier during ultrasonic treatment, preventing nucleic acid breakage caused by mechanical shearing force. This formula has been experimentally verified to effectively preserve nucleic acids at room temperature and prevent degradation.

[0052] (6) Stable composition: The nucleic acid release agent has a simple and stable composition, is not easily affected by external environmental factors, is easy to prepare and store, can be stored for a long time under normal temperature or refrigeration conditions, and has a low cost, which is conducive to large-scale production and promotion.

[0053] In summary, this invention, by developing an extraction-free rapid nucleic acid release agent and introducing an ultrasonic treatment scheme for rapid sample pretreatment, successfully solves the problems of cumbersome, time-consuming, and inefficient nucleic acid extraction processes in existing technologies, providing an efficient, convenient, and low-cost nucleic acid release and detection solution for clinical testing and scientific research. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0055] Figure 1 The fluorescence amplification curve of reagent 4, which is a nucleic acid release agent with added permeabilizer (DMSO) and metal chelating agent (EGTA), is shown.

[0056] Figure 2 The fluorescence amplification curves of the reagents prepared according to the concentration and ratio of Formula 11 are shown.

[0057] Figure 3 The image shows the fluorescence amplification curve of a 50 copies / mL oropharyngeal swab sample obtained by ultrasonic lysis of the nucleic acid release agent of this invention.

[0058] Figure 4 The image shows the fluorescence amplification curve of a 50 copies / mL sputum sample tested using the nucleic acid release agent of this invention via ultrasonic lysis.

[0059] Figure 5 The image shows the fluorescence amplification curve of a 50 copies / mL sample of bronchoalveolar lavage fluid tested using the nucleic acid release agent of this invention via ultrasonic lysis.

[0060] Figure 6 The image shows the fluorescence amplification curve of a 50 copies / mL serum sample detected by ultrasonic lysis of the nucleic acid release agent of the present invention.

[0061] Figure 7 The image shows the fluorescence amplification curve of a 50 copies / mL plasma sample detected by ultrasonic lysis of the nucleic acid release agent of the present invention.

[0062] Figure 8 The image shows the fluorescence amplification curve of a 50 copies / mL sample of viral culture lysed by ultrasonication using the nucleic acid release agent of this invention.

[0063] Figure 9 The image shows the fluorescence amplification curve of a 50 copies / mL whole blood sample detected by ultrasonic lysis of the nucleic acid release agent of the present invention.

[0064] Figure 10 The image shows the fluorescence amplification curve of a 50 copies / mL fecal sample detected by ultrasonic lysis of the nucleic acid release agent of this invention. Detailed Implementation

[0065] This invention discloses a nucleic acid releasing agent, its preparation method, and its application.

[0066] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0067] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0068] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0069] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0070] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0071] The technical solution provided by this invention includes:

[0072] The combined effect of physical lysis and chemical release: This invention primarily provides an extraction-free rapid nucleic acid release agent, its preparation method, and its application method, aiming to revolutionize the traditional nucleic acid extraction process and solve the core pain points commonly found in existing technologies, such as cumbersome operation steps, lengthy processing cycles, and low extraction efficiency. Traditional nucleic acid extraction methods typically require multiple complex steps, including cell lysis, protein removal, and nucleic acid purification. These methods not only rely on experimental equipment but also place stringent demands on the professional skills of operators, resulting in a generally time-consuming process. This invention innovatively constructs a rapid nucleic acid release system based on the combined effect of physical lysis and chemical release.

[0073] High-frequency ultrasonic treatment: Addressing the limitations of existing nucleic acid release agent methods in fully releasing sufficient nucleic acids from complex samples, this invention innovatively introduces a high-frequency ultrasonic treatment scheme as a key technology for sample pretreatment. Through the instantaneous high-temperature and high-pressure environment generated by cavitation, micron-sized bubbles are induced to periodically form and oscillate in the negative pressure phase of the acoustic waves, achieving nanoscale cell membrane disruption and homogenization of the sample. This non-contact physical lysis method offers significant advantages over traditional mechanical grinding or enzymatic digestion, including high lysis efficiency, short processing time, and superior protection of nucleic acid integrity. The specially designed combination of ultrasonic treatment parameters ensures effective lysis of various microorganisms and animal cells while avoiding nucleic acid chain breakage caused by excessive ultrasonication.

[0074] Extraction-Free Rapid Nucleic Acid Release Agent: This invention successfully developed a rapid nucleic acid release agent containing components such as dissociative salts, buffer solution, polyethylene glycol, surfactant, antifreeze, and metal chelating agent, through optimized synergistic ratios of each reagent component. The application of a novel nonionic surfactant significantly reduces the inhibitory effect on subsequent PCR detection while maintaining cell lysis efficiency; the added metal chelating agent effectively removes nuclease activity from the sample. Experimental verification shows that this release agent exhibits excellent applicability and efficiency for various complex sample types, including swabs, sputum, and blood.

[0075] A "One-Step" Operation Process: This invention combines an extraction-free rapid nucleic acid release agent with an ultrasonic treatment scheme, forming a highly efficient and convenient nucleic acid release process. Operators only need to mix the sample with the release agent and perform a short ultrasonic treatment before directly adding the lysate to the downstream detection system. The entire process eliminates the need for organic solvent extraction, magnetic bead purification, or centrifugation, reducing the time from sample processing to nucleic acid readiness to less than one minute. This provides a more efficient, convenient, and universal solution for multiple fields such as biomedical research, disease diagnosis, and epidemic prevention and control.

[0076] This invention provides an extraction-free rapid nucleic acid release agent, which can rapidly and efficiently release nucleic acids from samples, simplifying the nucleic acid detection process and improving detection efficiency. Based on this, this invention innovatively introduces an ultrasonic treatment scheme for rapid sample pretreatment, thereby improving the efficiency of nucleic acid release and enabling pretreatment for different sample types, further enhancing the practicality and application scope of this technology. Simultaneously, this invention also provides a method for preparing and using this nucleic acid release agent.

[0077] To achieve the above objectives, the technical solution of the present invention is as follows:

[0078] Components of the nucleic acid release agent:

[0079] Nucleic acid release agents are mainly composed of 1 mol / L to 50 mol / L dissociation salt, 5 mmol / L to 100 mmol / L buffer, 1% to 15% (w / v) polyethylene glycol, 0.01% to 1% (v / v) surfactant, and 1% to 15% (v / v) antifreeze.

[0080] Release salts include guanidine thiocyanate, guanidine hydrochloride, potassium thiocyanate, etc., with guanidine thiocyanate being preferred. The concentration is 1 mol / L to 50 mol / L. These salts have the ability to disrupt protein structure and the interaction between nucleic acids and proteins, thus facilitating the release of nucleic acids.

[0081] Buffer solutions include Tris-HCl buffer, sodium citrate buffer, etc., with Tris-HCl buffer being preferred. The concentration is 5 mmol / L to 100 mmol / L to maintain the pH stability of the system and provide a suitable environment for nucleic acid release.

[0082] Permeabilizers include methyl sulfoxide, dimethyl sulfoxide, decyl methyl sulfoxide, etc., with dimethyl sulfoxide being preferred. The concentration is 1% to 10% (v / v). They can penetrate the cell membrane, disrupt the membrane structure by changing the fluidity of membrane lipids, and promote the release of nucleic acids.

[0083] Polyethylene glycol: Polyethylene glycol-n, n=200~20000, preferably n=2000, concentration 1%-15% (w / v).

[0084] Surfactants include Triton X-100, sodium dodecyl sulfonate, CHAPS, etc., with Triton X-100 being preferred at a concentration of 0.01% to 1% (v / v). As a nonionic surfactant, it can disrupt cell membranes and viral envelopes, allowing nucleic acids to be released.

[0085] Antifreeze agents include ethylene glycol, propylene glycol, glycerol, etc., with glycerol being preferred. At a concentration of 1%–15%, these agents lower the freezing point of water, reducing ice crystal formation and thus protecting nucleic acids from ice crystal damage at low temperatures. They can also form a protective film around nucleic acid molecules, preventing them from contacting harmful substances and stabilizing the nucleic acid structure.

[0086] Metal chelating agents: including one or more of EGTA, EDTA, and DTPA, with EGTA being preferred, at a concentration of 1-10 mM. Metal chelating agents can chelate metal ions in the sample, preventing metal ions from interfering with the nucleic acid release process and subsequent PCR reactions, while also helping to maintain the stability of nucleic acids.

[0087] Antioxidants include one or more of vitamin C, glutathione, and tea polyphenols, with glutathione being preferred; the concentration of the antioxidant is 1 mmol / L to 5 mmol / L. The antioxidants scavenge free radicals, block oxidation reactions, and maintain the stability of nucleic acid structure and function.

[0088] pH value: Adjust the pH to 6-9, which is conducive to the release and stability of nucleic acids.

[0089] Preparation method:

[0090] 1. Weigh out guanidine thiocyanate and dissolve it in an appropriate amount of deionized water.

[0091] 2. Measure out the Tris-HCl buffer solution and add it to the above solution.

[0092] 3. Weigh out PEG2000 and add it to the above solution, then stir to dissolve it.

[0093] 4. Weigh out EGTA and glutathione and add them to the above solution, then stir to dissolve them.

[0094] 5. Measure out Triton-100, DMSO and glycerol and add them to the above solution, then stir thoroughly until homogeneous.

[0095] 6. Slowly adjust the pH of the mixed solution to 6-9 while stirring.

[0096] 7. Finally, dilute to 1000 mL with deionized water to obtain the extraction-free rapid nucleic acid release agent.

[0097] Ultrasonic treatment conditions: The present invention describes a sample lysis treatment combining ultrasound and chemical methods. The frequency of the ultrasound treatment is 10kHz to 100kHz, preferably 40 to 50kHz; the time is 1s to 60s, preferably 10s to 30s.

[0098] The unit of polyethylene glycol in this invention is g / mL.

[0099] In Examples 1 to 9 of this invention, all raw materials and reagents used can be purchased from the market.

[0100] The present invention will be further illustrated below with reference to the embodiments:

[0101] Example 1

[0102] Different component reagents were added to the formula of the nucleic acid release agent to prepare different formulas of nucleic acid release agent. The nucleic acid release agents of formula 1 to formula 4 were prepared according to Table 1 to Table 4 respectively. The positive sample of influenza A virus was diluted to 100 copies / mL as a positive sample. The nucleic acid was amplified and detected by repeatedly spotting 10 reactions using the respiratory tract 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) of Guangzhou Baochuang Biotechnology Co., Ltd. For specific operation steps, please refer to the kit instructions.

[0103] The formulation of the nucleic acid release agent is shown in Table 1.

[0104] Table 1 Nucleic Acid Release Agent Formulation 1

[0105]

[0106] The nucleic acid release agent formulation 2 is shown in Table 2.

[0107] Table 2 Nucleic Acid Release Agent Formulation 2

[0108]

[0109] The nucleic acid release agent formulation is shown in Table 3.

[0110] Table 3 Nucleic Acid Release Agent Formulation 3

[0111]

[0112] The formulation of the nucleic acid release agent is shown in Table 4.

[0113] Table 4 Nucleic Acid Release Agent Formulation 4

[0114]

[0115] Table 5. Test results of nucleic acid release agent formulations 1-4

[0116]

[0117] As shown in Table 5, the detection rate of formulation 1 is 0%, that of formulation 2 is 20%, that of formulation 3 is 30%, and that of formulation 4 is 100%. If the effects of DMSO and EGTA are independent (i.e., without synergistic effects), then the detection rate when used simultaneously can be calculated using a probabilistic independent action model. Specifically, the probability of successful detection should be equal to 1 minus the probability of both failing.

[0118] The data shows that without DMSO and EGTA, the detection failure probability is 100% (i.e., 1 - 0% = 1). With only DMSO, the detection failure probability is 80% (i.e., 1 - 20% = 0.8). With only EGTA, the detection failure probability is 70% (i.e., 1 - 30% = 0.7). Under the assumption of independent action, when using DMSO and EGTA simultaneously, the probability of detection failure is the product of the individual failure probabilities of each (because they are independent), that is: P(failure | DMSO and EGTA) = P(failure | DMSO) × P(failure | EGTA) = 0.8 × 0.7 = 0.56.

[0119] Therefore, the probability of successful detection (detection rate) is: P(success | DMSO and EGTA) = 1 - 0.56 = 0.44, which is 44%. Experimental results show that the detection rate of formulation 4 is 100%, far exceeding 44%, indicating a significant synergistic effect between the penetrant (DMSO) and the metal chelating agent (EGTA), which greatly improves the detection rate when used together. The detection results for formulation 4 are attached. Figure 1 As shown.

[0120] Example 2

[0121] Different component reagents were added to the nucleic acid release agent formulation to prepare different formulations of nucleic acid release agents. The formulations were prepared according to Tables 6 to 9 to obtain formulations 5 to 8. The positive samples of influenza A virus were diluted to 100 copies / mL as positive samples and placed at room temperature for 0, 1, 2, 3, 4, 5, 6 and 7 days. The nucleic acid was amplified and detected by repeatedly spotting 10 reactions using the respiratory tract 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. For specific operation steps, please refer to the kit instructions.

[0122] The formulation of the nucleic acid release agent is shown in Table 6.

[0123] Table 6 Nucleic Acid Release Agent Formulation 5

[0124]

[0125] The formulation of the nucleic acid release agent is shown in Table 7.

[0126] Table 7 Nucleic Acid Release Agent Formulation 6

[0127]

[0128] The formulation of the nucleic acid release agent is shown in Table 8.

[0129] Table 8 Nucleic Acid Release Agent Formulation 7

[0130]

[0131] The formulation of the nucleic acid release agent is shown in Table 9.

[0132] Table 9 Nucleic Acid Release Agent Formulation 8

[0133]

[0134] Table 10. Nucleic acid stability test after release of different nucleic acid release agents.

[0135]

[0136] Based on the data in Table 10, we can calculate the upper limit of the detection rate theoretically when glycerol and glutathione are added simultaneously without synergistic effect (i.e., their effects are independent). If the effects of glycerol and glutathione are independent, then the expected detection rate of Formula 8 can be calculated using a probability model: Expected detection rate = 1 - (1 - Detection rate of Formula 6) × (1 - Detection rate of Formula 7). This value represents the upper limit of the detection rate without synergistic effect.

[0137] Day 0 to Day 2: Detection rate of Formula 6 is 100%, and the detection rate of Formula 7 is 100%. Expected detection rate = 1 - (1-1) × (1-1) = 1 - 0 = 100%, with an upper limit of 100%.

[0138] Day 3: Detection rate of formula 6 is 80%, and detection rate of formula 7 is 70%. Expected detection rate = 1 - (1 - 0.8) × (1 - 0.7) = 1 - (0.2 × 0.3) = 1 - 0.06 = 94%, with an upper limit of 94%.

[0139] Day 4: Detection rate of formula 6 is 40%, and detection rate of formula 7 is 50%. Expected detection rate = 1 - (1 - 0.4) × (1 - 0.5) = 1 - (0.6 × 0.5) = 1 - 0.3 = 70%, with an upper limit of 70%.

[0140] Day 5: Formula 6 detection rate 10%, Formula 7 detection rate 20%. Expected detection rate = 1 - (1 - 0.1) × (1 - 0.2) = 1 - (0.9 × 0.8) = 1 - 0.72 = 28%, with an upper limit of 28%.

[0141] Day 6-7: Detection rate of formula 6 is 0%, and the detection rate of formula 7 is 0%. Expected detection rate = 1 - (1-0) × (1-0) = 1 - (1 × 1) = 0%, with an upper limit of 0%.

[0142] If glycerol and glutathione do not have a synergistic effect (the effects are independent), the upper limit of the detection rate of Formula 8 should be the calculated value above. The actual detection rate of Formula 8 was 100% from day 0 to day 7. Starting from day 3, the actual value (100%) was significantly higher than the upper limit under independent effects (94% on day 3, 70% on day 4, 28% on day 5, and 0% on days 6-7).

[0143] This indicates that the simultaneous addition of the nucleic acid stabilizer (glycerol) and the antioxidant (glutathione) produced a strong synergistic effect, significantly improving the stability of the nucleic acid releaser.

[0144] Example 3

[0145] In this embodiment, nucleic acid release agents were prepared according to different concentrations and proportions of the formulation components. The nucleic acid release agents of formulations 9 to 13 were prepared according to Tables 11 to 15. The positive samples of influenza A virus were diluted to 100 copies / mL as positive samples. The nucleic acid was amplified and detected by repeatedly spotting 10 reactions using the respiratory 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. For specific operation steps, please refer to the kit instructions.

[0146] Nucleic acid release agent formulation 9 is shown in Table 11.

[0147] Table 11 Nucleic Acid Release Agent Formulation 9

[0148]

[0149] Nucleic acid release agent formulation 10 is shown in Table 12.

[0150] Table 12 Nucleic Acid Release Agent Formulation 10

[0151]

[0152] Nucleic acid release agent formulation 11 is shown in Table 13.

[0153] Table 13 Nucleic Acid Release Agent Formulation 11

[0154]

[0155] Nucleic acid release agent formulation 12 is shown in Table 14.

[0156] Table 14 Nucleic Acid Release Agent Formulation 12

[0157]

[0158] Nucleic acid release agent formulation 13 is shown in Table 15.

[0159] Table 15 Nucleic Acid Release Agent Formulation 13

[0160]

[0161] Table 16 Test results of nucleic acid release agent formulations 1-4

[0162]

[0163] As shown in Table 16, formulations 10, 11, and 12 all achieved excellent detection results for nucleic acid release from samples, with a consistent 100% detection rate for positive samples at 100 copies / mL. However, formulations 9 and 13 were less efficient than formulations 10, 11, and 12. The detection results for formulation 11 are shown below. Figure 2 As shown.

[0164] In summary, the components of the rapid nucleic acid extraction reagent of the present invention are shown in Table 17 below. Further tests will be conducted using formulation 11.

[0165] Table 17 Formulation of the nucleic acid extraction or purification reagent of the present invention

[0166]

[0167] Example 4: Ultrasonic treatment to accelerate nucleic acid release

[0168] Different frequency tests: Sampling tubes containing epidermal mucosal cells scraped from the buccal mucosa were prepared and supplemented with nucleic acid release agent. The ultrasonic processor was set to continuous mode, with frequencies of 30 kHz, 40 kHz, and 50 kHz, and runtimes of 10 s, 30 s, 1 min, and 2 min, respectively. The sampling tubes were placed in the sample processing chamber of the ultrasonic processor, locked, and the ultrasonic program was started. After processing, the supernatant was collected for PCR detection.

[0169] Table 18 Exploration Tests under Different Ultrasonic Conditions

[0170]

[0171] The results in Table 18 show that 40-50 kHz and 10-30 s can significantly improve the release effect of nucleic acid, indicating that this frequency is more conducive to the efficient release of nucleic acid from the sample. In subsequent tests, 40 kHz and 10 s were selected.

[0172] Example 5: Comparison of results between commercially available nucleic acid release agents and conventional magnetic bead-based nucleic acid extraction reagents.

[0173] The nucleic acid release agent configured with the optimal components in Example 1 was used to dilute positive samples and compared with commercially available magnetic bead-based nucleic acid extraction reagents. The results showed that the nucleic acid release agent of the present invention was more effective.

[0174] Influenza A virus-positive samples were diluted to 2000 copies / mL, 1000 copies / mL, 500 copies / mL, 200 copies / mL, 100 copies / mL, 50 copies / mL, and 25 copies / mL respectively using negative oropharyngeal swab samples, sputum samples, bronchoalveolar lavage fluid samples, serum samples, plasma samples, virus culture samples, whole blood samples, and fecal samples. The samples were then added according to the following procedure. Ten replicates of extraction were performed using the nucleic acid release agent and control reagent of this invention to verify the results. Two groups of nucleic acid release agents were prepared: Group 1 was vortexed, and Group 2 was sonicated. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating steps were referred to the kit instructions. After extraction, amplification and detection were performed using the respiratory tract 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating steps were referred to the kit instructions.

[0175] 1. Sample addition:

[0176] Swab sample: Immediately insert the collected swab into the sampling tube containing the nucleic acid release agent, break it off near the top of the sampling tube and discard the tail, then tighten the cap of the sampling tube.

[0177] Sputum sample: Add 50-100 μL of sputum into a sampling tube containing nucleic acid release agent and tighten the cap.

[0178] Virus culture medium: Add 100-300 μL of virus culture medium sample to a sampling tube containing nucleic acid release agent and tighten the cap.

[0179] Serum and plasma: Take 100-300 μL of serum or plasma sample and add it to a sampling tube containing nucleic acid release agent, then tighten the cap.

[0180] For irrigation fluid and effusion samples: Take 100~300μL of irrigation fluid or effusion samples and add it to a sampling tube containing nucleic acid release agent, then tighten the cap.

[0181] Whole blood sample: Add 10-50 μL of whole blood sample to a sampling tube containing nucleic acid release agent and tighten the cap.

[0182] Fecal sample: Take 1-2g of fecal sample and add it to a sampling tube containing nucleic acid release agent, then tighten the cap.

[0183] 2. Sample processing:

[0184] Method 1: Vortex mixing

[0185] Place the nucleic acid release agent containing the sample on a mixer, vortex thoroughly for 15 seconds, let stand for 5 minutes, and then aspirate the supernatant for downstream PCR detection.

[0186] Treatment Method 2: Ultrasonic Decomposition

[0187] Ultrasonic processor parameter settings: Select continuous mode for operation, and set the frequency and running time.

[0188] Operating steps: Place the nucleic acid release agent containing the sample into the sample processing tank of the ultrasonic processor, ensure that it is stable after being placed away from the sample tube, lock the sample tube, start the ultrasonic processor, and continue until the set ultrasonic program is completed. The supernatant can be used for downstream PCR detection.

[0189] The results of ultrasonic lysis of oropharyngeal swab samples with the nucleic acid release agent of this invention (50 copies / mL) are as follows: Figure 3 As shown.

[0190] The results of ultrasonic lysis of sputum samples (50 copies / mL) using the nucleic acid release agent of this invention are as follows: Figure 4 As shown.

[0191] The results of the detection of 50 copies / mL of bronchoalveolar lavage fluid samples using the nucleic acid release agent of this invention are as follows: Figure 5 As shown.

[0192] The results of ultrasonic lysis of serum samples (50 copies / mL) using the nucleic acid release agent of this invention are as follows: Figure 6 As shown.

[0193] The results of the detection of 50 copies / mL plasma samples by ultrasonic lysis of the nucleic acid releasing agent of this invention are as follows: Figure 7 As shown.

[0194] The results of the detection of 50 copies / mL of viral culture samples by ultrasonic lysis of the nucleic acid release agent of this invention are as follows: Figure 8 As shown.

[0195] The nucleic acid releasing agent of this invention was used to ultrasonically lyse whole blood samples of 50 copies / mL. The results of the sample detection are as follows: Figure 9 As shown.

[0196] The results of ultrasonic lysis of 50 copies / mL fecal samples using the nucleic acid release agent of this invention are as follows: Figure 10 As shown.

[0197] Table 19 Extraction performance data of the nucleic acid releasing agent and control reagent of the present invention

[0198]

[0199] According to the data in Table 19, for different sample types, the detection limit of the nucleic acid release agent of the present invention is better than that of the control kits that are already on the market with 30-minute extraction.

[0200] Example 6: Interference of nucleic acid releasing agents on PCR reagents

[0201] The nucleic acid of the positive sample was serially diluted with the nucleic acid release agent and pure water prepared with the optimal components in Example 1. The diluted nucleic acid was then amplified and detected using the respiratory tract 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. The specific operation steps are as described in the kit instructions.

[0202] Table 20 Amplification efficiency of the nucleic acid release agent and pure water of the present invention on PCR reagents

[0203]

[0204] As shown in Table 20, there was no significant difference in the PCR amplification results when the nucleic acid samples were serially diluted with nucleic acid release agent and pure water, respectively, indicating that this nucleic acid release agent does not interfere with downstream PCR reagents.

[0205] Example 7: Extraction efficiency of nucleic acid release agent for different sample types

[0206] 1. The extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent for respiratory oropharyngeal swab samples:

[0207] Nucleic acid extraction validation from 16 respiratory oropharyngeal swab samples: Nucleic acid was extracted from 16 influenza A virus oropharyngeal swab samples using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent results were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the respiratory tract 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0208] Table 21 Performance data of the nucleic acid release agent of the present invention in oropharyngeal swab samples

[0209]

[0210] According to the data in Table 21, for nucleic acid extraction of 16 positive samples, comparing the nucleic acid extraction effect of the kit of the present invention and the control kit in oropharyngeal swab clinical samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit.

[0211] 2. For respiratory sputum samples, the extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent:

[0212] Nucleic acid extraction validation from 16 respiratory sputum samples: Nucleic acid was extracted from 16 Streptococcus pneumoniae samples using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent results were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the respiratory tract 15 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0213] Table 22 Performance data of the nucleic acid release agent of the present invention in sputum samples

[0214]

[0215] According to the data in Table 22, for nucleic acid extraction of 16 clinical samples, comparing the nucleic acid extraction effect of the kit of the present invention and the control kit in sputum clinical samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit.

[0216] 3. The extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent for bronchoalveolar lavage fluid samples:

[0217] Nucleic acid extraction validation from 16 bronchoalveolar lavage fluid samples: Nucleic acid extraction was performed on 16 Streptococcus pneumoniae samples using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent results were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the respiratory tract 15 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0218] Table 23 Performance data of the nucleic acid release agent of the present invention in sputum samples

[0219]

[0220] According to the data in Table 23, for nucleic acid extraction of 16 clinical samples, comparing the nucleic acid extraction effect of the kit of the present invention and the control kit in bronchoalveolar lavage fluid clinical samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit.

[0221] 4. For serum samples, the extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent:

[0222] Nucleic acid extraction and verification were performed on 16 serum samples from each of the following groups: Hepatitis B virus (HBV) serum samples were extracted using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the Hepatitis B Virus (HBV) Nucleic Acid Detection Kit (Fluorescent PCR Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0223] Table 24 Performance data of the nucleic acid release agent of the present invention in serum samples

[0224]

[0225] According to the data in Table 24, for nucleic acid extraction of 16 clinical samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit in serum samples compared with the control kit.

[0226] 5. For plasma samples, the extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent:

[0227] Nucleic acid extraction and validation were performed on 16 plasma samples from each of the following groups: Hepatitis B virus serum samples were extracted using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the Hepatitis B Virus (HBV) Nucleic Acid Detection Kit (Fluorescent PCR Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0228] Table 25 Performance data of the nucleic acid release agent of the present invention in plasma samples.

[0229]

[0230] According to the data in Table 25, for nucleic acid extraction from 16 clinical samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit in plasma samples compared with the control kit.

[0231] 6. The extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent for viral culture samples:

[0232] Nucleic acid extraction and verification were performed on 16 viral culture samples from each sample. Hepatitis B virus serum samples were extracted using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent results were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the Hepatitis B Virus (HBV) Nucleic Acid Detection Kit (Fluorescent PCR Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0233] Table 26 Performance data of the nucleic acid release agent of the present invention in virus culture samples

[0234]

[0235] According to the data in Table 26, for nucleic acid extraction of 16 clinical samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit in nucleic acid extraction of viral culture samples.

[0236] 7. For whole blood samples, the extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent:

[0237] Nucleic acid extraction verification was performed on 16 whole blood samples from each of the following groups: Hepatitis B virus serum samples were extracted using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent results were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the Hepatitis B Virus (HBV) Nucleic Acid Detection Kit (Fluorescent PCR Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0238] Table 27 Performance data of the nucleic acid release agent of the present invention in whole blood samples.

[0239]

[0240] According to the data in Table 27, for nucleic acid extraction from 16 clinical samples, comparing the nucleic acid release agent of the present invention with the control kit in whole blood samples, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit.

[0241] 8. For fecal samples, the extraction performance of the nucleic acid release agent of this invention was compared and verified with that of the control reagent:

[0242] Nucleic acid extraction and verification were performed on 16 whole blood samples from each of the following groups: Norovirus nucleic acid was extracted from the samples using a nucleic acid release agent following the aforementioned ultrasonic lysis procedure. Each clinical sample was extracted once, and negative and positive controls from the amplification kit were extracted once each. The detection rate and Ct value of the control reagent were compared. The control reagent was the nucleic acid extraction or purification kit (catalog number: BQ4012) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions. After extraction, amplification and detection were performed using the Human Infectious Diarrheal Virus Nucleic Acid Detection Kit (Fluorescent PCR-Melting Curve Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Specific operating procedures were followed according to the kit instructions.

[0243] Table 28 Performance data of the nucleic acid release agent of the present invention in fecal samples.

[0244]

[0245] According to the data in Table 28, for nucleic acid extraction from 16 clinical samples, comparing the nucleic acid release agent of the present invention with the control kit in fecal blood sample extraction, the nucleic acid release agent of the present invention has a better extraction efficiency than the control kit.

[0246] Example 8

[0247] I. Real-time stability test

[0248] The nucleic acid release agent of this invention was placed at room temperature and taken out at 0 months, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months and 24 months respectively. Positive samples were diluted with the nucleic acid release agent as a diluent to 500 copies / mL as positive samples (P1~P10), and blank nucleic acid release agent was used as negative samples (N1~N10). Ten samples were set up for each sample. After the viral nucleic acid was extracted, the respiratory 12 pathogen nucleic acid detection kit (fluorescent PCR melting curve method) of Guangzhou Baochuang Biotechnology Co., Ltd. was used for amplification and detection. The specific operation steps are referred to the kit instructions.

[0249] Table 29 Results of Real-Time Stability Tests for the Reagent Kit

[0250]

[0251] As shown in Table 29, after 24 months of storage at room temperature, the nucleic acid release agent of the present invention remained clear, without any precipitate or damage to its appearance. The positive and negative sample concordance rates were satisfactory, and the CV% of positive samples was no higher than 5%. All test results of the reagent kit were satisfactory, indicating that the reagent kit of the present invention has long-term stability when stored at room temperature.

[0252] II. Low Temperature Stability

[0253] The nucleic acid release agent of the present invention was placed in a refrigerator at 2~8°C, and diluted positive samples were taken out and tested at 0 months, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months and 24 months respectively (the specific operation is the same as the real-time stability test).

[0254] Table 30 Results of low-temperature stability test of the reagent kit

[0255]

[0256] As shown in Table 30, after the kit of Example 4 was stored at 2~8℃ for 24 months, the release agent solution was clear, without precipitate, and the appearance was undamaged. The positive and negative sample concordance rates were qualified, and the CV% of positive samples was not higher than 5%. All test results of the kit were qualified, indicating that the kit of the present invention has long-term stability when stored at room temperature.

[0257] III. Stability at 37℃

[0258] Place the kit in a 37°C incubator and remove diluted positive samples at 0, 1, 3, 6, 9, 12, 18, and 24 months for testing (the specific procedure is the same as for real-time stability testing).

[0259] Table 31 Results of reagent kit stability test at 37℃

[0260]

[0261] As shown in Table 31, after the kit of Example 4 was placed at 37°C for 24 months, the release agent solution was clear, without any precipitate, and the appearance was undamaged. The positive and negative sample concordance rates were qualified, and the CV% of positive samples was not higher than 5%. All test results of the kit were qualified, indicating that the kit of the present invention has long-term stability when stored at room temperature.

[0262] IV. Stability at 50℃

[0263] Place the kit in a 50°C incubator and remove diluted positive samples at 0, 1, 3, 6, 9, and 12 months for testing (the specific procedure is the same as for real-time stability testing).

[0264] Table 32 Results of reagent kit stability test at 37℃

[0265]

[0266] As shown in Table 32, after the kit of Example 4 was placed at 50°C for 24 months, the release agent solution was clear, without any precipitate, and the appearance was undamaged. The positive and negative sample concordance rates were qualified, and the CV% of positive samples was not higher than 5%. All test results of the kit were qualified, indicating that the kit of the present invention has long-term stability when stored at room temperature.

[0267] Example 9: Post-release nucleic acid stability test

[0268] Negative oropharyngeal swab samples, sputum samples, bronchoalveolar lavage fluid samples, serum samples, plasma samples, viral culture samples, whole blood samples, and stool samples were added to the nucleic acid release agent of this invention. Positive clinical samples were diluted to 50 copies / mL, and 10 replicates of extraction were performed for verification. The samples were incubated at room temperature for 0, 1, 2, 3, 4, 5, 6, and 7 days. Nucleic acid from oropharyngeal swab samples was amplified and detected using the Respiratory 12 Pathogen Nucleic Acid Detection Kit (Fluorescent PCR Melting Curve Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Nucleic acid from sputum and bronchoalveolar lavage fluid samples was amplified and detected using the Respiratory 15 Pathogen Nucleic Acid Detection Kit (Fluorescent PCR Melting Curve Method) from Guangzhou Baochuang Biotechnology Co., Ltd. Nucleic acid from serum, plasma, viral culture, and whole blood samples was detected using the Hepatitis B Virus (HBV) Nucleic Acid Detection Kit (Fluorescent PCR Method) from Guangzhou Baochuang Biotechnology Co., Ltd. For amplification and detection of human infectious diarrhea virus (HIV) samples, Guangzhou Baochuang Biotechnology Co., Ltd.'s Human Infectious Diarrhea Virus Nucleic Acid Detection Kit (Fluorescent PCR-Melting Curve Method) was used. For specific operating procedures, please refer to the kit instructions.

[0269] Table 33 Nucleic acid stability test after release of the nucleic acid release agent of the present invention

[0270]

[0271] According to the data in Table 33, for different sample types, the nucleic acid released by the nucleic acid release agent of the present invention can be stored at room temperature for 7 days.

[0272] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid releasing agent, characterized in that, The components include:

2. The nucleic acid release agent of claim 1, wherein, The chaotropic salt includes one or more of guanidine thiocyanate, guanidine hydrochloride and potassium thiocyanate.

3. The nucleic acid releasing agent of claim 1 or 2, wherein The surfactant includes one or more of Triton X-100, sodium dodecyl sulfate and CHAPS.

4. The nucleic acid release agent of any one of claims 1 to 3, wherein, The antifreezing agent includes one or more of ethylene glycol, propylene glycol and glycerol.

5. The nucleic acid release agent of any one of claims 1 to 4, wherein, The permeating agent includes one or more of methyl sulfoxide, dimethyl sulfoxide and decyl methyl sulfoxide.

6. The nucleic acid release agent of any one of claims 1 to 5, wherein, The metal chelator includes one or more of EGTA, EDTA and DTPA.

7. The nucleic acid release agent of any one of claims 1 to 6, wherein, The antioxidant includes one or more of vitamin C, glutathione and tea polyphenol.

8. The nucleic acid release agent of any one of claims 1 to 7, wherein, The buffer includes Tris-HCl buffer and / or sodium citrate buffer.

9. The preparation method of the nucleic acid releasing agent according to any one of claims 1 to 8, comprising the following steps: mixing the components, adjusting the pH value to obtain the nucleic acid releasing agent.

10. The nucleic acid releasing agent according to any one of claims 1 to 8 and / or the nucleic acid releasing agent obtained by the preparation method of claim 9 is used in any of the following: (a) nucleic acid extraction; and / or (b) preparation of the product of nucleic acid extraction; and / or (c) nucleic acid detection; and / or (d) preparation of the product of nucleic acid detection.

11. Use according to claim 10, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The nucleic acid extraction includes the following steps: mixing the sample to be tested with the nucleic acid releasing agent, and ultrasonicating to obtain the sample.

12. The use according to claim 11, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The frequency of the ultrasonicating is 10-100 KHz, and the time is 1-60 s.

13. A test reagent and / or test kit, characterized in that The components include: The nucleic acid releasing agent according to any one of claims 1 to 8 and / or the nucleic acid releasing agent obtained by the preparation method of claim 9 and acceptable adjuvants, carriers and / or devices.

14. The method of using a test agent and / or test kit according to claim 13, wherein, The detection reagent and / or the detection kit are mixed with the sample to be tested, and ultrasonicated.

15. The method of use of claim 14, wherein, The frequency of the ultrasonicating is 10-100 KHz, and the time is 1-60 s.

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