Preservation reagent for viscous biological samples, kit and use thereof

Abstract

The application relates to the field of in vitro diagnosis, in particular to a viscous biological sample preservation reagent, a kit and application thereof, further relates to a pretreatment method of the viscous biological sample, an amplification method and a detection method of nucleic acid in the viscous biological sample. The viscous biological sample preservation reagent comprises a liquefying agent, a nucleic acid protection agent, a surfactant and an aqueous solvent; the preservation reagent comprises guaifenesin with a concentration of 0.24 mg / mL-2.4 mg / mL and 0.01% (v / v)-0.1% (v / v) beta-mercaptoethanol as the liquefying agent. The viscous biological sample preservation reagent can reduce the viscosity of the viscous biological sample, realize direct amplification without extraction and ensure room temperature stability.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic technology, and in particular to reagents, kits and their applications for preserving viscous biological samples, and further to pretreatment methods for viscous biological samples, amplification methods for nucleic acids in viscous biological samples and detection methods. Background Technology

[0002] In the field of molecular diagnostics, especially in the detection of gynecological infectious diseases based on nucleic acid amplification technologies (such as PCR, isothermal amplification, etc.), gynecological samples such as vaginal secretions and cervical secretions are key test targets. These samples have complex compositions, typically containing a large number of exfoliated epithelial cells, white blood cells, microorganisms (such as bacteria, fungi, protozoa), and varying amounts of mucus (cervical mucus and vaginal secretions).

[0003] While current liquid media used for collecting and preserving such samples can maintain nucleic acid stability and inactivate pathogens to some extent, they exhibit the following serious drawbacks when handling highly viscous samples, severely impacting detection accuracy and operational efficiency:

[0004] 1. Poor pipetting accuracy and high risk of contamination: Viscous samples easily lead to inaccurate aspiration and incomplete drainage of liquid from the pipette tip, and are highly prone to "stringing" (or "stringing"). This phenomenon not only causes sampling volume errors, but also easily leads to sample contamination of the tube wall, cap, and surrounding environment, resulting in cross-contamination and aerosol risks. Current methods for liquefying viscous biological samples lack effective liquefaction capabilities and cannot effectively break down the viscous matrix of the sample, resulting in the aforementioned defects in volume errors, stringing contamination, and low nucleic acid release rates during pipetting operations.

[0005] 2. Sample heterogeneity and poor repeatability: Samples tend to form gel-like clumps in the preservation reagent, making it difficult to achieve uniform dispersion through vortexing. This results in large fluctuations in the number of pathogens in each aliquot of the sample during nucleic acid extraction, leading to poor repeatability of amplification and detection results, and a high risk of missing low-load pathogens.

[0006] 3. Insufficient pathogen release and limited sensitivity: The viscous mucus network forms a physical barrier, encapsulating pathogens and hindering the effective contact and action of lysis components, resulting in low nucleic acid release efficiency and ultimately affecting detection sensitivity, especially for detecting low-concentration infections.

[0007] 4. Existing biological sample testing solutions primarily rely on nucleic acid extraction, making it difficult to balance testing efficiency and accuracy. While commonly used nucleic acid extraction and detection methods can guarantee reliable results, they involve multiple steps such as lysis, binding, washing, and elution, resulting in cumbersome and time-consuming processes that cannot meet the urgent needs for rapid testing in community healthcare and outpatient settings.

[0008] In summary, existing viscous biological sample processing technologies have significant shortcomings in terms of sample liquefaction, detection sensitivity, and process efficiency, failing to meet the needs of rapid and accurate clinical testing. Therefore, there is an urgent need to develop a viscous biological sample pretreatment technology that can effectively break down viscous sample matrices, achieve rapid homogenization and liquefaction, and balance ease of operation with high detection sensitivity, thus filling the gaps in current technologies. Summary of the Invention

[0009] Therefore, it is necessary to provide a reagent, kit, and application for preserving viscous biological samples, as well as a pretreatment method for viscous biological samples, a method for amplifying nucleic acids in viscous biological samples, and a detection method.

[0010] In one aspect, some embodiments provide a preservation reagent for viscous biological samples, the preservation reagent comprising a liquefying agent, a nucleic acid protectant, a surfactant, and an aqueous solvent.

[0011] The liquefying agent comprises guaiacol ether and β-mercaptoethanol.

[0012] The concentration of guaiacol in the preservation reagent is 0.24 mg / mL to 2.4 mg / mL, and the concentration of β-mercaptoethanol is 0.01% (v / v) to 0.1% (v / v).

[0013] In an optional embodiment, the nucleic acid protectant comprises at least one of sucrose, glycerol, mannitol, and proline.

[0014] In an optional embodiment, the concentration of sucrose in the preservation reagent is 1 mg / mL to 10 mg / mL.

[0015] In an optional embodiment, the concentration of mannitol in the preservation reagent is 0.5 mg / mL to 5 mg / mL.

[0016] In an optional embodiment, the concentration of proline in the preservation reagent is 1 mg / mL to 10 mg / mL.

[0017] In an optional embodiment, the concentration of glycerol in the preservation reagent is 0.05% (v / v) to 0.5% (v / v).

[0018] In an optional embodiment, the preservation reagent comprises 1 mg / mL to 10 mg / mL of sucrose, 0.5 mg / mL to 5 mg / mL of mannitol, 1 mg / mL to 10 mg / mL of proline, and 0.05% (v / v) to 0.5% (v / v) of glycerol.

[0019] In an optional embodiment, the surfactant includes at least one of Triton X-100 and PVP40.

[0020] In an optional embodiment, the concentration of Triton X-100 in the preservation reagent is 0.002% (v / v) to 0.1% (v / v).

[0021] In an optional embodiment, the concentration of PVP40 in the preservation reagent is 0.02 mg / mL to 1 mg / mL.

[0022] In an optional embodiment, the preservation reagent comprises 0.002% (v / v) to 0.1% (v / v) of Triton X-100 and 0.02 mg / mL to 1 mg / mL of PVP40.

[0023] In an optional embodiment, the pH of the aqueous solvent is 8.0 to 8.5.

[0024] In an optional embodiment, the aqueous solvent is a buffer solution.

[0025] In an optional embodiment, the buffer solution includes at least one of Tris-HCl buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, and disodium hydrogen phosphate-citrate buffer.

[0026] In an optional embodiment, the buffer solution is a Tris-HCl buffer solution, and the buffer system for preserving the reagent is a Tris-HCl buffer system with a pH of 8.0-8.5 and a concentration of 50-100 mM.

[0027] In an optional embodiment, the preservation reagent comprises 0.24 mg / mL to 2.4 mg / mL guaiacol, 0.01% (v / v) to 0.1% (v / v) β-mercaptoethanol, 1 mg / mL to 10 mg / mL sucrose, 0.5 mg / mL to 5 mg / mL mannitol, 1 mg / mL to 10 mg / mL proline, 0.05% (v / v) to 0.5% (v / v) glycerol, 0.002% (v / v) to 0.1% (v / v) Triton X-100, and 0.02 mg / mL to 1 mg / mL PVP40, wherein the aqueous solvent is a 50-100 mM Tris-HCl buffer solution.

[0028] Secondly, some embodiments provide a kit for detecting viscous biological samples, the kit comprising the preservation reagent for viscous biological samples described in the first aspect.

[0029] In an optional implementation, the kit may further include nucleic acid detection reagents.

[0030] In an optional implementation, the nucleic acid detection reagent includes a nucleic acid amplification reagent.

[0031] Thirdly, some embodiments provide the application of the preservation reagent for viscous biological samples described in the first aspect, or the kit for detecting viscous biological samples described in the second aspect, in the preparation of products for detecting gynecological infectious diseases.

[0032] In an optional embodiment, the test sample in the gynecological infectious disease detection product includes the subject's reproductive tract secretions.

[0033] In an optional embodiment, the gynecological infectious disease detection product is used to detect nucleic acids in the viscous biological sample.

[0034] In an optional embodiment, the gynecological infectious disease detection product is used in a method comprising the steps of: mixing the viscous biological sample with the preservation reagent, and then using the resulting mixture directly for detection.

[0035] Fourthly, some embodiments provide a pretreatment method for viscous biological samples, the pretreatment method comprising mixing the viscous biological sample with a preservation reagent for viscous biological samples as described in the first aspect.

[0036] Fifthly, some embodiments provide a method for amplifying nucleic acids in a sticky biological sample, the amplification method comprising treating the sticky biological sample according to the pretreatment method for the sticky biological sample described in the fourth aspect, and performing amplification treatment on the resulting mixture to amplify the nucleic acids in the resulting sample;

[0037] In an optional embodiment, the viscous biological sample is mixed with the preservation reagent, and the resulting mixture is directly added to the amplification system for amplification reaction.

[0038] In a sixth aspect, some embodiments provide a method for detecting nucleic acids in viscous biological samples not intended for diagnosis or treatment, the method comprising processing the viscous biological sample according to the pretreatment method for viscous biological samples described in the fourth aspect, and detecting nucleic acids in the processed sample;

[0039] In an optional embodiment, the detection method includes amplifying the target nucleic acid using the nucleic acid amplification method for viscous biological samples described in the fifth aspect.

[0040] The preservation reagent for viscous biological samples provided in this application contains liquefying components that can degrade mucins and genomic DNA networks in the sample, reducing its viscosity and achieving fluidization and homogenization of the sample. This ensures accurate pipetting and reduces contamination, laying a reliable foundation for subsequent detection. The preservation reagent can release and stabilize nucleic acids during liquefaction, achieving long-term stable preservation of nucleic acids at room temperature. The preservation reagent of this application addresses three aspects: reducing the viscosity of viscous biological samples, simultaneously releasing and stabilizing nucleic acids, and ensuring room temperature stability. This achieves efficient sample liquefaction and reduced sample viscosity while guaranteeing efficient subsequent nucleic acid amplification. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0042] Figure 1 The amplification curves for different sample processing methods in Example 1 are shown.

[0043] Figure 2 The amplification curves are shown in Example 2 after treating viscous samples with different formulations. Detailed Implementation

[0044] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0046] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0047] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0048] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0049] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0050] In this application, the terms "biological sample," "sample," etc., refer to animal samples; tissues or organs, tissue lysates, or other biological samples derived from animals (preferably including at least mammals, such as primates, including humans); cells (in vivo of a subject, directly taken from the subject, or held in a culture or derived from a cultured cell line), cell lysates (or portions of lysates), or cell extracts; solutions containing one or more molecules derived from cells, cellular material, or viral material (e.g., polypeptides or nucleic acids); or solutions containing naturally occurring or non-naturally occurring nucleic acids, which are or can be measured as described herein. In some embodiments, the sample contains nucleic acids. The sample may also be any bodily fluid or excretion containing one or more cells, cellular components, or nucleic acids, including but not limited to cells, cell nuclei, or cell-free nucleic acids. In particular, the biological samples of this application are preferably derived from bodily fluids, including liquids, semi-solids, aerated liquids, liquid-gas mixtures, etc., derived from animals. Body fluids can be secretory or non-secretory, and exemplary body fluids include, but are not limited to, reproductive tract secretions (e.g., vaginal secretions, cervical secretions, and semen), saliva, sputum, serum, plasma, blood, urine, mucus, sweat, tears or other eye fluids, ear fluids, gastric juice or gastric juice, fecal fluid, pancreatic juice or juice, semen, breast milk, cerebrospinal fluid, fluid bone marrow, or lymph.

[0051] In this application, "viscous biological sample" refers to a biological sample that is viscous, particularly viscous bodily fluids. The viscosity may originate from the presence of abundant mucins and polysaccharides (especially mucopolysaccharides) or proteoglycans in the biological sample. Optionally, the viscous biological sample includes reproductive tract secretions, such as vaginal secretions or cervical secretions; more preferably, the viscous biological sample includes cervical mucus.

[0052] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0053] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0054] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0055] In one aspect, some embodiments provide a preservation reagent for viscous biological samples, the preservation reagent comprising a liquefying agent, a nucleic acid protectant, a surfactant, and an aqueous solvent.

[0056] In this preservation reagent, the liquefaction agent refers to the component that can reduce the viscosity of viscous biological samples. The liquefaction agent includes guaiacol and β-mercaptoethanol. The concentration of guaiacol in the preservation reagent is 0.24 mg / mL to 2.4 mg / mL, for example, but not limited to 0.24 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.2 mg / mL or 2.4 mg / mL; the concentration of β-mercaptoethanol is 0.01% (v / v) to 0.1% (v / v), for example, but not limited to 0.01% (v / v), 0.02% (v / v), 0.03% (v / v), 0.04% (v / v), 0.05% (v / v), 0.06% (v / v), 0.07% (v / v), 0.08% (v / v), 0.09% (v / v) or 0.1% (v / v). Guaifenesin and β-mercaptoethanol can degrade mucins and genomic DNA networks in samples, reducing sample viscosity.

[0057] In this preservation reagent, the nucleic acid protectant refers to the agent that stabilizes nucleic acid molecules released by pathogens in viscous biological samples, maintaining their integrity and thus ensuring the detection sensitivity and effectiveness of subsequent PCR amplification. In optional embodiments, the nucleic acid protectant includes at least one of sucrose, glycerol, mannitol, and proline.

[0058] In an optional embodiment, the concentration of sucrose in the preservation reagent is 1 mg / mL to 10 mg / mL, for example, but not limited to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL.

[0059] In an optional embodiment, the concentration of mannitol in the preservation reagent is 0.5 mg / mL to 5 mg / mL, for example, but not limited to 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0060] In an optional embodiment, the concentration of proline in the preservation reagent is 1 mg / mL to 10 mg / mL, for example, but not limited to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL.

[0061] In an optional embodiment, the concentration of glycerol in the preservation reagent is 0.05% (v / v) to 0.5% (v / v), for example, but not limited to, 0.05% (v / v), 0.1% (v / v), 0.15% (v / v), 0.2% (v / v), 0.25% (v / v), 0.3% (v / v), 0.35% (v / v), 0.4% (v / v), 0.45% (v / v), or 0.5% (v / v).

[0062] In an optional embodiment, the preservation reagent comprises 1 mg / mL to 10 mg / mL of sucrose, 0.5 mg / mL to 5 mg / mL of mannitol, 1 mg / mL to 10 mg / mL of proline, and 0.05% (v / v) to 0.5% (v / v) of glycerol.

[0063] In an optional embodiment, the surfactant includes at least one of Triton X-100 and PVP40.

[0064] In an optional embodiment, the concentration of Triton X-100 in the preservation reagent is 0.002% (v / v) to 0.1% (v / v), for example, but not limited to, 0.002% (v / v), 0.005% (v / v), 0.01% (v / v), 0.02% (v / v), 0.03% (v / v), 0.04% (v / v), 0.05% (v / v), 0.06% (v / v), 0.07% (v / v), 0.08% (v / v), 0.09% (v / v), or 0.1% (v / v).

[0065] In an optional embodiment, the concentration of PVP40 in the preservation reagent is 0.02 mg / mL to 1 mg / mL, for example, but not limited to, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL.

[0066] In an optional embodiment, the preservation reagent comprises 0.002% (v / v) to 0.1% (v / v) of Triton X-100 and 0.02 mg / mL to 1 mg / mL of PVP40.

[0067] In an optional embodiment, the pH of the aqueous solvent is 8.0 to 8.5, such as, but not limited to, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5.

[0068] In an optional embodiment, the aqueous solvent is a buffer solution. Further optionally, the buffer solution includes, but is not limited to, at least one of Tris-HCl buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, and disodium hydrogen phosphate-citrate buffer.

[0069] In an optional embodiment, the aqueous solvent is a Tris-HCl buffer solution. The Tris-HCl buffer solution provides a buffer system of Tris-HCl with a pH of 8.0 to 8.5 and a concentration of 50 to 100 mM for preserving the reagent. The concentration of Tris-HCl in the buffer system for preserving the reagent is, for example, but not limited to, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0070] In an optional embodiment, the preservation reagent comprises a liquefying agent, a nucleic acid protectant, and a surfactant, with the remainder being an aqueous solvent.

[0071] In an optional embodiment, the preservation reagent comprises 0.24 mg / mL to 2.4 mg / mL guaiacol, 0.01% (v / v) to 0.1% (v / v) β-mercaptoethanol, 1 mg / mL to 10 mg / mL sucrose, 0.5 mg / mL to 5 mg / mL mannitol, 1 mg / mL to 10 mg / mL proline, 0.05% (v / v) to 0.5% (v / v) glycerol, 0.002% (v / v) to 0.1% (v / v) Triton X-100 and 0.02 mg / mL to 1 mg / mL PVP40, and Tris-HCl buffer at pH 8.0 to 8.5 and a concentration of 50 to 100 mM.

[0072] In an optional embodiment, the preservation reagent comprises 0.24 mg / mL to 2.4 mg / mL guaiacol, 0.01% (v / v) to 0.1% (v / v) β-mercaptoethanol, 1 mg / mL to 10 mg / mL sucrose, 0.5 mg / mL to 5 mg / mL mannitol, 1 mg / mL to 10 mg / mL proline, 0.05% (v / v) to 0.5% (v / v) glycerol, 0.002% (v / v) to 0.1% (v / v) Triton X-100, and 0.02 mg / mL to 1 mg / mL PVP40, with the balance being Tris-HCl buffer at pH 8.0 to 8.5 and a concentration of 50 to 100 mM.

[0073] This application reveals that using guaiacol and β-mercaptoethanol as the main liquefaction components in a preservation reagent for viscous biological samples can effectively reduce the viscosity of these samples. When nucleic acids are not extracted from the viscous biological samples, and the preservation reagent used to treat the viscous biological samples is directly used as the sample in the nucleic acid amplification reaction system, the preservation reagent using guaiacol and β-mercaptoethanol as liquefaction agents can achieve good amplification results, with amplification efficiency close to that obtained by using magnetic beads to extract nucleic acids as the amplification sample. Good detection precision and sensitivity can also be obtained. Furthermore, guaiacol and β-mercaptoethanol exhibit a synergistic effect. When guaiacol is used alone or in combination with DTT (dithiothreitol), both the liquefaction effect and amplification efficiency of viscous biological samples are reduced. When β-mercaptoethanol is combined with other liquefaction components (N-acetylcysteine), the amplification efficiency is reduced. Without adding a liquefaction agent or using other combinations of liquefaction agents (such as bromhexine hydrochloride and TCEP), good direct amplification results cannot be obtained.

[0074] In some embodiments, the preservation reagent contains a liquefying component that degrades mucins and genomic DNA networks in the sample, reducing its viscosity, achieving fluidization and homogenization of the sample, ensuring accurate pipetting, reducing contamination, and laying a reliable foundation for subsequent detection. This preservation reagent releases and stabilizes nucleic acids during liquefaction, inhibits DNase and RNase activity, neutralizes nucleic acid amplification inhibitors, and allows for direct amplification without the need for nucleic acid extraction and purification steps, achieving long-term stable preservation of nucleic acids at room temperature.

[0075] The preservation reagents in some embodiments address three aspects: reducing the viscosity of viscous biological samples, enabling extraction-free direct amplification, and ensuring room temperature stability. While achieving efficient sample liquefaction and reducing viscosity issues, they eliminate the need for nucleic acid extraction and purification steps, allowing direct subsequent nucleic acid amplification and detection, and can protect the integrity of sample nucleic acids for a short period at room temperature. These preservation reagents possess significant advantages such as high liquefaction efficiency, good direct amplification compatibility, room temperature stability, and simplified operation.

[0076] Secondly, some embodiments provide a kit for detecting viscous biological samples, the kit comprising a preservation reagent for viscous biological samples as described in the first aspect, the kit being used to preserve viscous biological samples, placing the viscous biological samples in the preservation reagent can reduce the viscosity of the viscous biological samples, and subsequently they can be directly used for nucleic acid detection without the need for extraction and purification of nucleic acids in the preservation reagent, the nucleic acid detection including but not limited to the detection of at least one of DNA and RNA.

[0077] In an optional implementation, the kit may further include nucleic acid detection reagents, exemplary of which include, but are not limited to, at least one of nucleic acid amplification reagents, sequencing reagents, nucleic acid capture reagents, library construction reagents, chromatographic separation reagents, mass spectrometry detection reagents, CRISPR gene editing detection reagents, and reverse transcription reagents.

[0078] In an optional embodiment, the nucleic acid detection reagent includes a nucleic acid amplification reagent. Exemplary nucleic acid amplification reagents include, but are not limited to, at least one of enzymes, primers, probes, dNTPs, buffer components, and essential ionic components used for nucleic acid amplification reactions.

[0079] In an optional implementation, the kit contains reagents for performing at least one of the following nucleic acid amplification reactions: polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), strand displacement amplification (SDA), and rolling circle amplification (RCA).

[0080] In an optional implementation, the kit includes at least one of a negative control, a positive control, a standard, and a quality control.

[0081] Thirdly, some embodiments provide the application of the preservation reagent for viscous biological samples of the first aspect, or the kit for detecting viscous biological samples of the second aspect, in the preparation of gynecological infectious disease detection products. This preservation reagent or kit can be used as a sample storage component of the gynecological infectious disease detection product, for directly placing gynecological infectious disease samples, such as reproductive tract secretions, to reduce the viscosity of the viscous biological sample. Subsequently, the mixture of the viscous biological sample and the preservation reagent can be directly used for subsequent nucleic acid detection, such as direct nucleic acid amplification. When the kit also contains a nucleic acid detection reagent, the kit can be used as the nucleic acid detection component of the gynecological infectious disease detection product, where the nucleic acid to be detected includes, but is not limited to, at least one of DNA and RNA.

[0082] In an optional implementation, the test sample in the gynecological infectious disease detection product includes the subject's reproductive tract secretions.

[0083] In an optional implementation, the gynecological infectious disease detection product is used to detect nucleic acids in viscous biological samples.

[0084] In an optional implementation, the gynecological infectious disease detection product is used to detect pathogen nucleic acids in viscous biological samples.

[0085] In an optional embodiment, the gynecological infectious disease detection product is used in a method comprising the steps of: mixing a viscous biological sample with a preservation reagent, and then directly using the resulting mixture for detection.

[0086] Fourthly, some embodiments provide a pretreatment method for viscous biological samples, the pretreatment method comprising mixing the viscous biological sample with a preservation reagent for viscous biological samples according to the first aspect.

[0087] In an optional embodiment, the pretreatment method includes mixing the viscous biological sample with a preservation reagent and then inverting or shaking to mix. The mixing time is optionally 10s to 120s. In an optional embodiment, the mixing includes inverting to mix for 60 to 120s; in an optional embodiment, shaking to mix for 10 to 60s.

[0088] In an optional implementation, the pretreatment method for the viscous biological sample is not for diagnostic or therapeutic purposes.

[0089] Fifthly, some embodiments provide a method for amplifying nucleic acids in a sticky biological sample, the amplification method comprising treating the sticky biological sample according to the pretreatment method of the fourth aspect, performing amplification treatment on the resulting mixture to amplify nucleic acids in the resulting sample, the amplification comprising amplifying at least one of DNA and RNA.

[0090] In an optional embodiment, the amplification method includes mixing a viscous biological sample with a preservation reagent, and directly adding the resulting mixture to an amplification system for amplification reaction. That is, the system is directly amplified after mixing the viscous biological sample with the preservation reagent, without further extraction and purification of the nucleic acids therein.

[0091] In an optional implementation, the method for amplifying nucleic acids in the viscous biological sample is not for diagnostic or therapeutic purposes.

[0092] Sixthly, some embodiments provide a method for detecting nucleic acids in a sticky biological sample, the method comprising treating the sticky biological sample according to the pretreatment method for sticky biological samples in the fourth aspect, and detecting nucleic acids in the treated sample. Detection of nucleic acids includes, but is not limited to, detection of at least one of DNA and RNA. Nucleic acid detection can be performed using conventional methods known in the art, such as, but not limited to, nucleic acid amplification technologies (polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), cross-primer amplification (CPA), strand displacement amplification (SDA), and rolling circle amplification (RCA)), nucleic acid sequencing technologies (such as Sanger sequencing, next-generation high-throughput sequencing (NGS), nanopore sequencing, single-molecule real-time sequencing), nucleic acid hybridization technologies (such as nucleic acid probe hybridization, gene chips, microarray chips, fluorescence in situ hybridization FISH), CRISPR-Cas system-mediated nucleic acid detection, nucleic acid mass spectrometry analysis, electrochemical detection, biosensor detection, or immunochromatography.

[0093] In an optional implementation, the nucleic acid detection method includes amplifying the target nucleic acid using the nucleic acid amplification method in viscous biological samples according to the fifth aspect.

[0094] In an optional implementation, the detection method directly uses the mixture of viscous biological sample and preservation reagent after pretreatment by the fourth aspect as the detection sample. This detection method does not require extraction of nucleic acid from the viscous sample; the sample can be directly amplified or detected after liquefaction and preservation, which is beneficial for large-scale sample processing.

[0095] In an optional implementation, the method for detecting nucleic acids in the viscous biological sample is for diagnostic and therapeutic purposes. By detecting pathogens or subject nucleic acids in the viscous biological sample, it is possible to determine or assist in determining whether the subject is infected with pathogens, or to determine or assist in determining whether the subject has target symptoms or diseases.

[0096] In an optional implementation, the method for detecting nucleic acids in the viscous biological sample is not for diagnostic or therapeutic purposes.

[0097] Examples of non-diagnostic and non-therapeutic purposes mentioned above include, but are not limited to, obtaining nucleic acid information from viscous biological samples to obtain mutation information, transcriptome information, and pathogen abundance of the samples, and such nucleic acid information is not used for the diagnosis and treatment of diseases, such as for scientific research, biobank construction, and public health surveillance.

[0098] The following are some examples.

[0099] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0100] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0101] Example 1

[0102] A preservation reagent for viscous biological samples, comprising a liquefying agent, a nucleic acid protectant, a surfactant, and a buffer solution.

[0103] The liquefaction agent consists of guaiacol and β-mercaptoethanol, and the preservation reagent contains 0.24 mg / mL guaiacol and 0.01% (v / v) β-mercaptoethanol.

[0104] The nucleic acid protectant consists of sucrose, mannitol, glycerol and proline. The preservation reagent contains 1 mg / mL sucrose, 0.5 mg / mL mannitol, 1 mg / mL proline and 0.05% (v / v) glycerol.

[0105] The surfactant consists of Triton X-100 and PVP40, and the preservation reagent contains 0.002% (v / v) Triton X-100 and 0.02 mg / mL PVP40;

[0106] The buffer solution was pH 8.0 and had a concentration of 80 mM Tris-HCl.

[0107] The preparation method of this preservation reagent is as follows:

[0108] (a) Add 2.4 g of guaiacol and 1 mL of β-mercaptoethanol to sterile water and bring the volume to 10 mL to prepare a liquefaction agent for later use.

[0109] (b) Add 1g sucrose, 0.5g mannitol, 1g proline and 0.5mL glycerol to sterile water and bring the volume to 10mL to prepare a nucleic acid protectant for later use.

[0110] (c) Add 2g PVP40 and 2mL TritonX-100 to sterile water and make up to 10mL to prepare a surfactant for later use.

[0111] (d) The liquefaction agent, nucleic acid protectant and surfactant were mixed with buffer solution according to the addition amount of 0.1% (v / v) liquefaction agent, 1% (v / v) nucleic acid protectant and 0.01% (v / v) surfactant to prepare the preservation reagent.

[0112] Example 2

[0113] A preservation reagent for viscous biological samples, comprising a liquefying agent, a nucleic acid protectant, a surfactant, and a buffer solution.

[0114] The liquefaction agent consists of guaiacol and β-mercaptoethanol, and the preservation reagent contains 2.4 mg / mL guaiacol and 0.1% (v / v) β-mercaptoethanol.

[0115] The nucleic acid protectant consists of sucrose, mannitol, glycerol and proline. The preservation reagent contains 1 mg / mL sucrose, 0.5 mg / mL mannitol, 1 mg / mL proline and 0.05% (v / v) glycerol.

[0116] The surfactant consists of Triton X-100 and PVP40, and the preservation reagent contains 0.1% (v / v) Triton X-100 and 1 mg / mL PVP40;

[0117] The buffer solution was 100 mM Tris-HCl at pH 8.5.

[0118] The preparation method of this preservation reagent is as follows:

[0119] Steps (a) to (c) are the same as in Example 1.

[0120] (d) The liquefaction agent, nucleic acid protectant and surfactant were mixed with buffer solution according to the addition amount of 1% (v / v) liquefaction agent, 1% (v / v) nucleic acid protectant and 0.5% (v / v) surfactant to prepare the preservation reagent.

[0121] Example 3

[0122] A preservation reagent for viscous biological samples, comprising a liquefying agent, a nucleic acid protectant, a surfactant, and a buffer solution.

[0123] The liquefaction agent consists of guaiacol and β-mercaptoethanol, and the preservation reagent contains 0.24 mg / mL guaiacol and 0.01% (v / v) β-mercaptoethanol.

[0124] The nucleic acid protectant consists of sucrose, mannitol, glycerol and proline. The preservation reagent contains 10 mg / mL sucrose, 5 mg / mL mannitol, 10 mg / mL proline and 0.5% (v / v) glycerol.

[0125] The surfactant consists of Triton X-100 and PVP40, and the preservation reagent contains 0.002% (v / v) Triton X-100 and 0.02 mg / mL PVP40;

[0126] The buffer solution was pH 8.0 and had a concentration of 50 mM Tris-HCl.

[0127] The preparation method of this preservation reagent is as follows:

[0128] Steps (a) to (c) are the same as in Example 1.

[0129] (d) The liquefaction agent, nucleic acid protectant and surfactant were mixed with buffer solution according to the addition amount of 0.1% (v / v) liquefaction agent, 10% (v / v) nucleic acid protectant and 0.01% (v / v) surfactant to prepare the preservation reagent.

[0130] Example 4

[0131] A preservation reagent for viscous biological samples, comprising a liquefying agent, a nucleic acid protectant, a surfactant, and a buffer solution.

[0132] The liquefaction agent consists of guaiacol and β-mercaptoethanol. The preservation reagent contains 2.4 mg / mL guaiacol and 0.01% (v / v) β-mercaptoethanol.

[0133] The nucleic acid protectant consists of sucrose, mannitol, glycerol, and proline. The protectant contains 1 mg / mL sucrose, 0.5 mg / mL mannitol, 1 mg / mL proline, and 0.05% (v / v) glycerol.

[0134] The surfactant consists of Triton X-100 and PVP40. The preservation reagent contains 0.002% (v / v) Triton X-100 and 0.02 mg / mL PVP40;

[0135] The buffer solution was 100 mM Tris-HCl at pH 8.0.

[0136] The preparation method of this preservation reagent is as follows:

[0137] (a) Add 2.4 g of guaiac glycerol ether and 0.1 mL of β-mercaptoethanol to sterile water and bring the volume to 10 mL to prepare a liquefaction agent for later use.

[0138] (b) Add 1 g sucrose, 0.5 g mannitol, 1 g proline and 0.5 mL glycerol to sterile water and bring the volume to 10 mL to prepare a nucleic acid protectant for later use.

[0139] (c) Add 2 g PVP40 and 2 mL Triton X-100 to sterile water and bring the volume to 10 mL to prepare a surfactant for later use.

[0140] (d) The liquefaction agent, nucleic acid protectant and surfactant were mixed with buffer solution according to the addition amount of 1% (v / v) liquefaction agent, 1% (v / v) nucleic acid protectant and 0.01% (v / v) surfactant to prepare the preservation reagent.

[0141] Comparative Example 1

[0142] A preservation reagent, differing from Example 1 only in that it does not contain a liquefying agent.

[0143] Comparative Example 2

[0144] A preservation reagent differs from Example 1 only in that the liquefaction agent consists of guaiacol and DTT (dithiothreitol), and the preservation reagent contains 0.24 mg / mL guaiacol and 0.15 mg / mL DTT.

[0145] The preparation method of this preservation reagent is as follows:

[0146] (a) Add 2.4 g of guaiacol and 1.5 g of DTT to sterile water and make up to 10 mL to prepare a liquefaction agent for later use. The remaining steps are the same as in Example 1.

[0147] Comparative Example 3

[0148] A preservation reagent, differing from Example 1 only in that the liquefying agent contains only guaiacol ether.

[0149] The preparation method of this preservation reagent is as follows:

[0150] (a) Add 2.4 g of guaiacol to sterile water and make up to 10 mL to prepare a liquefaction agent for later use. The remaining steps are the same as in Example 1.

[0151] Comparative Example 4

[0152] A preservation reagent differs from Example 1 only in that the liquefaction agent consists of N-acetylcysteine ​​and β-mercaptoethanol, and the preservation reagent contains 0.5 mg / mL of N-acetylcysteine ​​and 0.01% (v / v) of β-mercaptoethanol.

[0153] The preparation method of this preservation reagent is as follows:

[0154] (a) Add 5g of N-acetylcysteine ​​and 1mL of β-mercaptoethanol to sterile water, and make up to 10mL to prepare a liquefying agent for later use. The remaining steps are the same as in Example 1.

[0155] Comparative Example 5

[0156] A preservation reagent differs from Example 1 only in that the liquefaction agent consists of bromhexine hydrochloride and TCEP (tris(2-carboxyethyl)phosphine), and the preservation reagent contains 0.5 mg / mL of bromhexine hydrochloride and 0.25 mg / mL of TCEP.

[0157] The preparation method of this preservation reagent is as follows:

[0158] (a) Add 5 g of bromhexine hydrochloride and 2.5 g of TCEP to sterile water and make up to 10 mL to prepare a liquefying agent for later use. The remaining steps are the same as in Example 1.

[0159] Comparative Example 6

[0160] A preservation reagent differs from Example 1 only in that the liquefaction agent is composed of DTT (dithiothreitol), and the preservation reagent contains 0.15 mg / mL of DTT.

[0161] The preparation method of this preservation reagent is as follows:

[0162] (a) Add 1.5 g DTT to sterile water and make up to 10 mL to prepare a liquefaction agent for later use. The remaining steps are the same as in Example 1.

[0163] Example 1

[0164] Direct amplification effect test

[0165] (1) Prepare preservation reagents 1 to 7 according to the six formulations of Example 1 and Comparative Examples 1 to 6 above. Soak clean cervical swabs that have not been sampled into 3 mL of preservation reagents 1, 2, 3, 4, 5, 6 and 7 respectively, tighten the cap, and mix by inverting for 90 seconds to simulate the real sampling environment.

[0166] (2) Using the seven preservation reagents in (1), the real samples of other types of human papillomavirus collected by TE were diluted 10 times to prepare seven preservation solution samples. These seven preservation solution samples are extraction-free direct amplification samples.

[0167] (3) Take another real sample of other types of human papillomavirus collected by TE in step (2), dilute it 10 times with TE, and take 1 mL of the diluted sample for extraction using a nucleic acid extraction and purification kit (model S10015 (16T), Sansure Biotech Inc.). Finally, use 60 μL of elution buffer to elute the viral nucleic acid for subsequent RT-PCR detection.

[0168] (4) The human papillomavirus nucleic acid detection kit of Sansure Biotech Inc. was used for detection. For specific operation details, please refer to the instruction manual. The reaction system is 20 μL of the sample obtained in step (2) or 5 μL of the sample obtained in step (3) by magnetic bead extraction + 40 μL of PCR reaction solution. The sample was then detected on the SLAN-96P fully automated medical PCR analysis system.

[0169] The results are as follows Figure 1 As shown, all of the above-mentioned preservation solutions 1-7 can be directly amplified without additional nucleic acid extraction steps. The results indicate that the combined liquefaction solution of guaiacol and β-mercaptoethanol in Example 1 has a Ct value close to that of the magnetic bead method (Ct cycle threshold, which refers to the number of cycles required for the fluorescence signal in the PCR reaction tube to reach a set threshold; a higher Ct value indicates a lower target nucleic acid content or lower nucleic acid amplification efficiency). It also exhibits higher fluorescence values ​​and is significantly superior to single-component guaiacol and other commonly used liquefaction solutions.

[0170] Example 2

[0171] Liquefaction effect and direct diffusion test

[0172] (1) Prepare preservation reagents 1 to 4 according to the four formulas of Example 1 and Comparative Examples 1 to 3 above. Take 1 mL of each of the four preservation solutions into a gynecological dropper.

[0173] (2) After mixing the collected viscous gynecological reproductive tract real samples, divide them into 5 equal parts. Add 4 of the samples to the preservation reagents 1 to 4 in step (1) to make viscous samples. Add 1 sample to 1 mL of TE solution.

[0174] (3) After shaking the viscous sample for 30 seconds, let it stand for 2 minutes, then press the dropper and count the time from pressing to complete dripping. Repeat each preservation solution 5 times.

[0175] (4) Take another 1 mL of TE sample from (2) and extract it using the nucleic acid extraction and purification reagent S10015 from Sansure Biotech Inc. For specific operation, refer to the instruction manual. Finally, use 60 μL of elution buffer to elute the viral nucleic acid for subsequent RT-PCR detection.

[0176] (5) The remaining samples from step (3) and the extracted samples from step (4) were tested using the human papillomavirus nucleic acid detection kit from Sansure Biotech Inc. For specific operation details, please refer to the instruction manual. The reaction system is 20 μL of the sample that was not extracted in step (3) or 5 μL of the nucleic acid extracted by magnetic bead method in step (4) + 40 μL of PCR reaction solution. The detection was performed on the SLAN-96P fully automated medical PCR analysis system.

[0177] The sample dripping time statistics are shown in Table 1 below: Formula 2 showed no obvious liquefaction effect, and the dripped liquid was stringy and could not drip quickly from the dropper; Formulas 1, 3, and 4 all showed obvious liquefaction effects, but the stringy effect was weakened, and the overall dripping time of the samples was between 1.5 and 2.5 seconds. The liquefaction effect of Formula 1 was better than that of Formulas 3 and 4.

[0178] The results of direct amplification of the viscous sample after liquefaction of the preservation solution are as follows: Figure 2 As shown: the Ct value of formulation 2 is significantly delayed, indicating a lower amplification efficiency. The Ct value of formulation 1, which has the best liquefaction effect, is earlier than that of formulations 2, 3, and 4, indicating a higher amplification efficiency. The amplification effect is close to that of the magnetic bead method, indicating that the combination of guaiacol and β-mercaptoethanol is superior to other combinations of liquefaction solutions.

[0179] Table 1. Sample dripping time after treating viscous samples with different formulations.

[0180]

[0181] Example 3

[0182] Preservative Precision Test

[0183] (1) Prepare preservation reagents No. 1 and No. 2 according to the formulations of Example 1 and Comparative Example 1 above. Soak the clean cervical swabs that have not been sampled into 3 mL of preservation reagent, tighten the cap, and mix by inverting for 90 seconds to simulate the real sampling environment.

[0184] (2) Use the preservation reagent in (1) to dilute the real viscous sample of other types of human papillomavirus collected by TE by 10 times to prepare the sample to be tested.

[0185] (3) The human papillomavirus nucleic acid detection kit of Sansure Biotech Inc. was used for detection. For specific operation details, please refer to the instruction manual. The reaction system is 20 μL of the sample to be tested + 40 μL of PCR reaction solution. Each group is repeated 8 times. The detection is performed on the SLAN-96P fully automated medical PCR analysis system.

[0186] The precision Ct values ​​of Example 1 and Comparative Example 1 are shown in the table below: The Ct value of Example 1 is significantly earlier than that of Example 1, indicating that the nucleic acid amplification efficiency is higher and the inter-well repeatability is significantly better than that of Comparative Example 1, with a coefficient of variation of less than 0.5%.

[0187] Table 2. Statistical analysis of Ct values ​​from precision tests in Example 1 and Comparative Example 1.

[0188]

[0189] Example of effect 4

[0190] Preservative solution sensitivity test

[0191] (1) Prepare preservation reagents No. 1 and No. 2 according to the formulations of Example 1 and Comparative Example 1 above. Soak the clean cervical swabs that have not been sampled into 3 mL of preservation reagent, tighten the cap, and mix by inverting for 90 seconds to simulate the real sampling environment.

[0192] (2) Use the preservation reagent from (1) to store 1×10 5 Other types of human papillomavirus plasmids were diluted to 400 copies / mL to prepare sensitive samples for subsequent detection.

[0193] (3) The human papillomavirus nucleic acid detection kit of Sansure Biotech Inc. was used for detection. For specific operation details, please refer to the instruction manual. The reaction system is 20 μL of the sample to be tested + 40 μL of PCR reaction solution. Each group is repeated 16 times and the detection is performed on the SLAN-96P fully automated medical PCR analysis system.

[0194] The sensitivity Ct values ​​of Example 1 and Comparative Example 1 are shown in the table below: Example 1 had a detection rate of 100% at the sensitivity detection concentration of the human papillomavirus nucleic acid detection kit, while Comparative Example 1 had a false negative.

[0195] Table 3. Statistical analysis of Ct values ​​from sensitivity tests in Example 1 and Comparative Example 1

[0196]

[0197] Example 5

[0198] Comparative test of different concentrations of components in the preservation solution

[0199] (1) Prepare preservation reagents 1 to 4 according to the four formulas in Examples 1 to 4 above.

[0200] Immerse clean, unsampled cervical swabs into 3 mL of preservation reagents 1, 2, 3, and 4 respectively, tighten the caps, and mix by inverting the tubes for 90 seconds to simulate a real sampling environment.

[0201] (2) Using the four preservation reagents in (1), the real samples of other types of human papillomavirus collected by TE were diluted 10 times to prepare four preservation solution samples. These four preservation solution samples are extraction-free direct amplification samples.

[0202] (3) Take another real sample of other types of human papillomavirus collected using the TE in step (2), dilute it 10 times with TE, and prepare a sample. Take 1 mL of the diluted sample and extract it using the nucleic acid extraction and purification reagent S10015 from Sansure Biotech Inc. For specific operation, refer to the instruction manual. Finally, use 60 μL of elution buffer to elute the viral nucleic acid for subsequent RT-PCR detection.

[0203] (4) The human papillomavirus nucleic acid detection kit of Sansure Biotech Inc. was used for detection. For specific operation details, please refer to the instruction manual. The reaction system is 20 μL of the sample obtained in step (2) or 5 μL of the sample obtained in step (3) by magnetic bead extraction + 40 μL of PCR reaction solution. Each group was repeated twice and the detection was performed on the SLAN-96P fully automated medical PCR analysis system.

[0204] As shown in Table 4, all four preservation solutions can be directly amplified without the need for additional nucleic acid extraction. Furthermore, the effects of the four preservation solutions are similar.

[0205] Table 4. Optimization Test of Different Component Concentrations in Preservative Solution - Ct Value Statistics

[0206]

[0207] Example 6

[0208] Stability test of preservation solution samples

[0209] (1) Prepare the preservation reagent according to the formula of Example 1 above.

[0210] (2) Dilute the 8 other types of human papillomavirus real viscous samples collected by TE with the preservation reagent in (1) by 10 times to prepare test samples 1 to 8. Store at room temperature.

[0211] (3) The human papillomavirus nucleic acid detection kit of Sansure Biotech Co., Ltd. was used for detection at regular intervals. For specific operation details, please refer to the instruction manual. The reaction system is 20 μL of the sample to be tested + 40 μL of PCR reaction solution. Each group was repeated twice and the detection was performed on the SLAN-96P fully automated medical PCR analysis system.

[0212] The results are shown in the table below. After 3 months of storage at room temperature, the Ct values ​​of the 8 samples changed by less than 1 from day 0, indicating that the preservation solution has a stable sample preservation function.

[0213] Table 5. Statistical analysis of Ct values ​​in the room temperature stability test of the preservation solution samples.

[0214]

[0215] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0216] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A reagent for preserving viscous biological samples, characterized in that, It contains liquefying agents, nucleic acid protecting agents, surfactants, and aqueous solvents; The liquefying agent comprises guaiac glycerol ether and β-mercaptoethanol; The concentration of guaiacol in the preservation reagent is 0.24 mg / mL to 2.4 mg / mL, and the concentration of β-mercaptoethanol is 0.01% (v / v) to 0.1% (v / v).

2. The preservation reagent for viscous biological samples according to claim 1, characterized in that, The nucleic acid protectant contains at least one of sucrose, glycerol, mannitol, and proline; Optionally, the concentration of sucrose in the preservation reagent is 1 mg / mL to 10 mg / mL; Optionally, the concentration of mannitol in the preservation reagent is 0.5 mg / mL to 5 mg / mL; Optionally, the concentration of proline in the preservation reagent is 1 mg / mL to 10 mg / mL; Optionally, the concentration of glycerol in the preservation reagent is 0.05% (v / v) to 0.5% (v / v). Optionally, the preservation reagent comprises 1 mg / mL to 10 mg / mL of sucrose, 0.5 mg / mL to 5 mg / mL of mannitol, 1 mg / mL to 10 mg / mL of proline, and 0.05% (v / v) to 0.5% (v / v) of glycerol.

3. The preservation reagent for viscous biological samples according to claim 1, characterized in that, The surfactant includes at least one of Triton X-100 and PVP40; Optionally, the concentration of Triton X-100 in the preservation reagent is 0.002% (v / v) to 0.1% (v / v). Optionally, the concentration of PVP40 in the preservation reagent is 0.02 mg / mL to 1 mg / mL; Optionally, the preservation reagent comprises 0.002% (v / v) to 0.1% (v / v) of Triton X-100 and 0.02 mg / mL to 1 mg / mL of PVP40.

4. The preservation reagent for viscous biological samples according to claim 1, characterized in that, The pH of the aqueous solvent is 8.0~8.5; Optionally, the aqueous solvent is a buffer solution; Optionally, the buffer solution includes at least one of Tris-HCl buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, and disodium hydrogen phosphate-citrate buffer. Optionally, the buffer solution is a Tris-HCl buffer solution, and the buffer system for preserving the reagent is a Tris-HCl buffer system with a pH of 8.0-8.5 and a concentration of 50-100 mM.

5. The preservation reagent for viscous biological samples according to any one of claims 1 to 4, characterized in that, The preservation reagent comprises 0.24 mg / mL to 2.4 mg / mL guaiacol, 0.01% (v / v) to 0.1% (v / v) β-mercaptoethanol, 1 mg / mL to 10 mg / mL sucrose, 0.5 mg / mL to 5 mg / mL mannitol, 1 mg / mL to 10 mg / mL proline, 0.05% (v / v) to 0.5% (v / v) glycerol, 0.002% (v / v) to 0.1% (v / v) Triton X-100, and 0.02 mg / mL to 1 mg / mL PVP40, and the aqueous solvent is a 50-100 mM Tris-HCl buffer.

6. A kit for detecting viscous biological samples, characterized in that, The kit contains the preservation reagent for viscous biological samples as described in any one of claims 1 to 5; Optionally, the kit may also include nucleic acid detection reagents; Optionally, the nucleic acid detection reagent includes a nucleic acid amplification reagent.

7. The use of the preservation reagent for viscous biological samples according to any one of claims 1 to 5, or the kit for detecting viscous biological samples according to claim 6, in the preparation of products for detecting gynecological infectious diseases; Optionally, the test sample in the gynecological infectious disease detection product includes the subject's reproductive tract secretions; Optionally, the gynecological infectious disease detection product is used to detect nucleic acids in the viscous biological sample; Optionally, the gynecological infectious disease detection product is used in a method comprising the steps of: mixing the viscous biological sample with the preservation reagent, and then using the resulting mixture directly for detection.

8. A pretreatment method for viscous biological samples, characterized in that, This includes mixing the viscous biological sample with the preservation reagent for viscous biological samples as described in any one of claims 1 to 5.

9. A method for amplifying nucleic acids in a viscous biological sample, characterized in that, The method includes processing the viscous biological sample according to the pretreatment method for viscous biological samples as described in claim 8, and performing amplification treatment on the resulting mixture to amplify the nucleic acids in the resulting sample; Optionally, the viscous biological sample is mixed with the preservation reagent, and the resulting mixture is directly added to the amplification system for amplification reaction.

10. A method for detecting nucleic acids in viscous biological samples for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes processing the viscous biological sample according to the pretreatment method for viscous biological samples as described in claim 8, and detecting nucleic acids in the processed sample; Optionally, the detection method includes amplifying the target nucleic acid using the nucleic acid amplification method for viscous biological samples as described in claim 9.

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