Liquefied combination product of viscous biological sample, nucleic acid detection kit and application thereof

Abstract

The invention provides a liquefaction combination product of a viscous biological sample, a nucleic acid detection kit and application of the liquefaction combination product. The liquefied combination product of the viscous biological sample comprises a first component and a second component, the first component is prepared from the following components: 10 mmol / L to 100 mmol / L of acetylcysteine, 5 mmol / L to 50 mmol / L of guaifenesin and 2 mmol / L to 20 mmol / L of 2-morpholine ethanesulfonic acid; the second component comprises a strong alkali solution, and the concentration of strong alkali in the strong alkali solution is 5 mol / L-20 mol / L. Specific acetylcysteine, guaifenesin and 2-morpholinoethanesulfonic acid are matched for use according to a specific proportioning relation, so that a viscous biological sample is quickly and sufficiently liquefied at room temperature, and the repeatability is relatively high. And the sample treated by the first component and the second component of the liquefied combined product prevents the strong acidity in the first component from interfering with the subsequent nucleic acid detection result.

Description

Technical Field

[0001] This application relates to the field of nucleic acid extraction and detection technology, and in particular to liquefaction combination products for viscous biological samples, nucleic acid detection kits and their applications. Background Technology

[0002] Viscous biological samples such as sputum, mucus, and tissue homogenates, as internal biological fluids, contain rich biomolecular information and are of great significance for the diagnosis and treatment of clinical diseases. In the field of nucleic acid detection, due to their high content of glycoproteins, mucopolysaccharides, and other viscous matrices, they are not only difficult to liquefy and disperse rapidly, but their complex components also tend to strongly inhibit subsequent nucleic acid amplification, becoming a core bottleneck restricting detection efficiency and accuracy.

[0003] Traditional nucleic acid testing relies on a separate process of "nucleic acid extraction and purification - amplification and detection." Its core logic involves obtaining high-purity nucleic acids through methods such as centrifugation, column chromatography, or magnetic bead adsorption, and then adding the purified nucleic acids to the amplification system for detection. The advantage of this approach is that the purification process effectively removes interfering substances such as proteins, polysaccharides, and enzyme inhibitors from the sample, creating a low-inhibition, low-interference, pure environment in the amplification system, thus ensuring the specificity and stability of the amplification reaction. However, this method has inherent drawbacks: the operation steps are cumbersome and time-consuming; extracting a single sample using magnetic beads alone takes more than 30 minutes, and the extraction process is prone to loss of the target nucleic acid or cross-contamination.

[0004] To simplify the process, one-step amplification technology has emerged, such as the liquefaction agent scheme for one-step amplification disclosed in invention patent application CN119709723A. The technical principle involves pre-mixing the reaction solution and enzyme in the amplification reagent to form a liquid reaction mix system, followed by directly adding a small amount of liquefied, unpurified sample for nucleic acid amplification. This scheme shortens the detection cycle by eliminating the nucleic acid extraction step and avoids the risk of nucleic acid loss and contamination during extraction. However, the sample after one-step processing still contains a large amount of PCR reaction inhibitors. The inhibitors are only diluted after adding the liquid reaction mix system to avoid affecting amplification. From the perspective of anti-interference mechanisms, one-step amplification relies on a single inhibitory enzyme or a simple buffer system for adjustment, and can only be used with liquid reagents and a small amount of sample for amplification. If combined with lyophilized reagents for full-sample amplification, the large amount of residual inhibitors from the one-step process directly enters the amplification system because the sample has not been purified, significantly affecting amplification efficiency. In summary, the liquefaction of traditional viscous biological samples has a significant inhibitory effect on subsequent lyophilized nucleic acid detection.

[0005] Therefore, traditional technologies still need improvement. Summary of the Invention

[0006] Based on this, this application provides a liquefaction combination product for viscous biological samples, a nucleic acid detection kit, and their applications. The liquefaction component in the liquefaction combination product can liquefy viscous biological samples simply, quickly, and efficiently, and can be adapted to lyophilized direct amplification systems and synergistically resist whole-sample amplification inhibitors.

[0007] The technical solution to the above-mentioned technical problems in this application is as follows:

[0008] A first aspect of this application provides a liquefaction combination product for viscous biological samples, comprising a first component and a second component;

[0009] The first component comprises: 10 mmol / L to 100 mmol / L acetylcysteine, 5 mmol / L to 50 mmol / L guaiacol, and 2 mmol / L to 20 mmol / L 2-morpholinoethanesulfonic acid;

[0010] The second component includes a strong alkali solution, wherein the concentration of the strong alkali in the strong alkali solution is 5 mol / L to 20 mol / L.

[0011] The aforementioned liquefaction product contains specific components and their specific ratios, including acetylcysteine, guaiacol, and 2-morpholinoethanesulfonic acid. Acetylcysteine ​​interacts with the disulfide bonds of mucin through its sulfhydryl groups, thereby cleaving the mucin molecules and also having a certain cleavage effect on DNA fibers, thus reducing sputum viscosity. 2-morpholinoethanesulfonic acid acts as a pH buffer, stabilizing the system's pH and maintaining the stability of nucleic acids in the liquefied solution. Guaifenesin also has a certain liquefaction effect on sputum in vitro. The specific ratio of acetylcysteine, guaiacol, and 2-morpholinoethanesulfonic acid allows for faster and more complete liquefaction of viscous biological samples at room temperature, with high reproducibility. Furthermore, samples treated with the combined first and second components of the above liquefaction product avoid interference from the strong acidity of the first component with subsequent nucleic acid detection results.

[0012] In some embodiments, the liquefied combination product satisfies at least one of the following characteristics:

[0013] (1) The concentration of acetylcysteine ​​is 50 mmol / L to 100 mmol / L;

[0014] (2) The concentration of the guaiacol is 20 mmol / L to 50 mmol / L;

[0015] (3) The concentration of the 2-morpholinoethanesulfonic acid is 5 mmol / L to 20 mmol / L;

[0016] (4) The strong base includes one or more of sodium hydroxide and potassium hydroxide.

[0017] In some embodiments, the viscous biological sample includes one or more of sputum, cervical mucus, nasopharyngeal swabs, and oral swabs.

[0018] A second aspect of this application provides the use of the liquefaction combination product of the aforementioned viscous biological sample in the preparation of a nucleic acid detection kit.

[0019] A third aspect of this application provides a nucleic acid detection kit, comprising a liquefied combination product of the aforementioned viscous biological sample.

[0020] In some embodiments, the nucleic acid detection kit further includes nucleic acid detection reagents, which include one or more of primers, probes, PCR buffer, DNA polymerase, UDG enzyme, and dNTPs.

[0021] A fourth aspect of this application provides a pretreatment method for viscous biological samples, comprising the following steps:

[0022] The liquefaction combination product of the viscous biological sample is used to liquefy the viscous biological sample to prepare a liquefied product.

[0023] In some embodiments, the step of liquefying the viscous biological sample includes the following steps:

[0024] The viscous biological sample is mixed with the first component and rigid particles in the liquefied composite product to prepare a first mixture;

[0025] The first mixture is mixed with a strong alkaline solution, and the pH of the system is adjusted to 8-10 to prepare a second mixture; and the second mixture is subjected to ultrasonic treatment to prepare the liquefied product.

[0026] In some embodiments, the pretreatment method for the viscous biological samples satisfies one or more of the following conditions:

[0027] (1) The rigid particles include one or more of zirconium oxide beads, zirconium silicate, glass beads, and stainless steel beads;

[0028] (2) The particle size of the rigid particles is 2 mm to 4 mm;

[0029] (3) The amount of the rigid particles used is 5 to 20; and,

[0030] (4) The frequency of the ultrasonic treatment is 10kHz~80kHz and the time is 60s~180s.

[0031] A fifth aspect of this application provides a method for detecting nucleic acids in viscous biological samples for non-diagnostic and non-therapeutic purposes, comprising the following steps:

[0032] Provide viscous biological samples;

[0033] The viscous biological sample is liquefied using the liquefaction combination product for the viscous biological sample as described above to prepare a liquefied product; and,

[0034] The target nucleic acid in the liquefied material is detected using nucleic acid detection reagents. Attached Figure Description

[0035] Figure 1 A comparison of stability and amplification curves for nucleic acid detection of sputum samples containing Streptococcus pneumoniae treated with the liquefied composition products of experimental groups 1-6 in Example 2;

[0036] Figure 2 A comparison of stability and amplification curves for nucleic acid detection of sputum samples containing Haemophilus influenzae treated with the liquefied composition products of experimental groups 1-6 in Example 2;

[0037] Figure 3 This is a comparison of the stability and amplification curves of nucleic acid detection in sputum samples containing Acinetobacter baumannii processed using the liquefied composition products of experimental groups 1-6 in Example 2. Detailed Implementation

[0038] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0039] 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 is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0040] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0041] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0042] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0043] The terms “first,” “second,” “third,” etc., are used for distinguishing 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.

[0044] In this application, concentration values ​​are defined as those within a certain range of fluctuation. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require extremely fine control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, or ±5%. Regarding molecular weight, fluctuations of ±10% are allowed.

[0045] In this application, the concentration or amount of each component in the liquefaction combination product, unless otherwise defined, refers to the final concentration or amount in the liquefaction combination product, which is equivalent to the final concentration or amount in the liquefaction reagent of the liquefaction combination product.

[0046] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0047] "Viscous biological samples" refers to biological samples that are viscous, particularly viscous bodily fluids. Viscosity can originate from the presence of abundant mucins and polysaccharides (especially mucopolysaccharides) or proteoglycans in the biological sample. A preferred viscous biological sample is sputum and / or cervical mucus. Examples of viscous biological samples include nasopharyngeal swabs, oral swabs, and lavage fluids. "Mucin" refers to any viscous protein that increases the viscosity of the cytoplasm surrounding secretory cells. "Sputum" refers to viscous material contained in or expelled from the nasal or oral cavity of mammals (usually expelled from the respiratory tract).

[0048] The term "release" of nucleic acids refers to the release of nucleic acids from a sample into a state where they are extractable, enriched, purified, and detectable. Nucleic acid release is usually accompanied by cell lysis and the physical separation of impurity components such as proteins, lipids, and polysaccharides from the nucleic acid components. Unlike in the physiological state, these components can be separated by simple methods such as centrifugation, and the nucleic acids can be directly detected by certain diagnostic reagents.

[0049] In this application, the term "strong base" preferably refers to, but is not limited to, a substance that ionizes entirely in aqueous solution as hydroxide ions. Unless otherwise specified, the OH radical used to provide alkalinity in a "strong base" can be 100% ionized into hydroxide ions in aqueous solution. The strong base referred to in this application can be an organic strong base or an inorganic strong base.

[0050] As shown in the background section, another traditional approach is the combined extraction and amplification using magnetic beads. For example, the liquefaction composition disclosed in patent application CN115704022A for magnetic bead extraction involves first liquefying the viscous sample with a liquefaction solution to disrupt its viscous structure and release nucleic acids. Then, the specific adsorption of magnetic beads in a high-salt environment captures the nucleic acids. After washing and elution, purified nucleic acids are obtained for subsequent amplification. The key technical focus of this approach is the step-by-step separation of liquefaction and extraction. The core function of the liquefaction solution is to assist nucleic acid release, not to adapt it to the subsequent amplification system. It removes inhibitors through the physical separation of the magnetic beads, ensuring the purity of the amplification system. However, this approach still has significant limitations: firstly, magnetic bead extraction requires multiple steps, including liquefaction, lysis, adsorption, washing, and elution, making the operation complex and difficult to automate quickly. The liquefaction process itself takes 15-30 minutes, resulting in a long overall process time. Secondly, the magnetic bead adsorption and elution processes may still cause nucleic acid loss, affecting the detection rate of low-concentration target nucleic acids.

[0051] Through long-term research, the applicant discovered that traditional one-step amplification, which uses a liquid system, and magnetic bead amplification, which requires extraction and separation before amplification, face the following core challenges: First, rapid and efficient liquefaction of viscous samples is required, which necessitates the destruction of the viscous matrix structure and the release of the target nucleic acid within a short time. Second, the compatibility of the lyophilized system must be ensured, so that after the liquefied sample (containing residual matrix and inhibitors) is reconstituted with the lyophilized amplification reagent, all components (enzymes, primers, probes, etc.) can still maintain their activity. Third, the interference resistance of whole-sample amplification is crucial, as the sample is not purified, and a large number of inhibitors directly enter the amplification system, resulting in a much higher interference complexity than traditional amplification after purification and one-step amplification.

[0052] Specifically, traditional post-purification amplification relies on physical separation to remove inhibitors, resulting in low interference but a cumbersome process; one-step amplification eliminates the need for purification, but the limitations of the liquid system make it difficult to handle the complex interferences of full-sample amplification; magnetic bead extraction, through liquefaction-assisted nucleic acid separation, can obtain pure nucleic acids, but the process is lengthy. The core technical challenge of lyophilized direct amplification technology lies in how to achieve rapid liquefaction of viscous samples without physical separation steps, while simultaneously overcoming the strong inhibitory interference from full-sample amplification through system co-design.

[0053] In summary, the liquefaction of traditional viscous biological samples generally suffers from problems such as complex operation and long time consumption. In addition, the high viscosity of the samples means that using them in conjunction with nucleic acid detection technology after liquefaction will interfere with subsequent nucleic acid detection.

[0054] Based on this, one embodiment of this application provides a liquefaction combination product for viscous biological samples, comprising a first component and a second component; the first component comprises: 10 mmol / L to 100 mmol / L acetylcysteine, 5 mmol / L to 50 mmol / L guaiacol and 2 mmol / L to 20 mmol / L 2-morpholinoethanesulfonic acid; the second component comprises a strong alkaline solution, wherein the concentration of the strong alkaline solution is 5 mol / L to 20 mol / L.

[0055] The aforementioned liquefaction product contains specific components and their specific ratios. Acetylcysteine, through the exchange of disulfide bonds between its sulfhydryl groups and mucin, cleaves mucin molecules and also has a certain cleavage effect on deoxyribonucleic acid fibers, thereby reducing sputum viscosity. 2-Molarin ethanesulfonic acid, as a pH buffer, stabilizes the system's pH value and maintains the stability of nucleic acids in the liquefaction solution. Guaifenesin also has a certain liquefaction effect on sputum in vitro. The specific ratio of acetylcysteine, guaiacyl ether, and 2-morpholino ethanesulfonic acid allows for faster and more complete liquefaction of viscous biological samples at room temperature, with high reproducibility. Furthermore, samples treated with the combined first and second components of the above liquefaction product avoid interference from the strong acidity in the first component with subsequent nucleic acid detection results.

[0056] In this application, the first component and the second component should, in principle, be packaged separately. The components of the first component may be packaged independently or in combination in one or more containers, and then mixed before use; in some preferred embodiments, the first component is packaged as a mixture in one container; the components of the second component may be packaged independently or in combination in one container.

[0057] It should be noted that the concentration of acetylcysteine ​​ranges from 10 mmol / L to 100 mmol / L, that is, the minimum and maximum values ​​within the range of 10 mmol / L to 100 mmol / L, as well as every value between these two values. Specific examples include, but are not limited to, the point values ​​in the examples and the following point values: 10 mmol / L, 12 mmol / L, 14 mmol / L, 16 mmol / L, 18 mmol / L, 20 mmol / L, 22 mmol / L, 24 mmol / L, 26 mmol / L, 28 mmol / L, 30 mmol / L, 32 mmol / L, 34 mmol / L, 36 mmol / L, 38 mmol / L, 40 mmol / L, 42 mmol / L, 44 mmol / L, 46 mmol / L, 48 mmol / L, 50 mmol / L, 52 mmol / L, 54 mmol / L, 56 mmol / L, 58 mmol / L, 60 mmol / L, 62 mmol / L, 64 mmol / L, 66 mmol / L, 68 mmol / L, 70 mmol / L, 72 mmol / L, 74 mmol / L, 76 mmol / L, 78 mmol / L, 80 mmol / L, 82 mmol / L, 84 mmol / L, 86 mmol / L, 88 mmol / L, 90 mmol / L, 92 mmol / L, 94 mmol / L, 96 mmol / L, 98 mmol / L, or 100 mmol / L, or a range of any two of these values, including, for example, 10 mmol / L to 80 mmol / L.

[0058] In some embodiments, the concentration of acetylcysteine ​​in the above-described liquefied combination product is 50 mmol / L to 100 mmol / L.

[0059] Understandably, controlling the concentration of acetylcysteine ​​within the above range results in better liquefaction of viscous biological samples.

[0060] The concentration range of guaiacol is "5 mmol / L to 50 mmol / L", which includes the minimum and maximum values ​​within this range, as well as every value between these values. Specific examples include, but are not limited to, the point values ​​in the examples and the following point values: 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L. mol / L, 30 mmol / L, 31 mmol / L, 32 mmol / L, 33 mmol / L, 34 mmol / L, 35 mmol / L, 36 mmol / L, 37 mmol / L, 38 mmol / L, 39 mmol / L, 40 mmol / L, 41 mmol / L, 42 mmol / L, 43 mmol / L, 44 mmol / L, 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, or 50 mmol / L, or any range of two of these values, for example, including: 5 mmol / L to 40 mmol / L.

[0061] In some embodiments, the concentration of the guaiacol ether in the above-described liquefied combination product is 20 mmol / L to 50 mmol / L.

[0062] Understandably, controlling the concentration of guaiacol within the above range results in better liquefaction of viscous biological samples.

[0063] The concentration of 2-morpholinoethanesulfonic acid ranges from 2 mmol / L to 20 mmol / L, meaning it can be the minimum and maximum value within this range, as well as every value between these values. Specific examples include, but are not limited to, the point values ​​in the examples and the following point values: 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, or 20 mmol / L, or any range consisting of any two of these values. For example, it includes 5 mmol / L to 15 mmol / L.

[0064] In some embodiments, the concentration of the 2-morpholinoethanesulfonic acid in the above-described liquefied combination product is 5 mmol / L to 20 mmol / L.

[0065] In some embodiments, the concentration of the strong base in the strong base solution as the second component is 5 mol / L to 20 mol / L. For example, it can be 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L, 10 mol / L, 11 mol / L, 12 mol / L, 13 mol / L, 14 mol / L, 15 mol / L, 16 mol / L, 17 mol / L, 18 mol / L, 19 mol / L, or 20 mol / L.

[0066] In this application, unless otherwise specified, the primary solvent in the liquefied combination product is water. The water referred to in this application can be distilled water, purified water, filtered water, deionized water, sterile water, etc. In some embodiments, the primary solvent in the liquefied combination product is sterile water. Unless otherwise specified, the water referred to in this application is preferably nucleic acid-free and nuclease-free. In some embodiments, the water referred to in this application may also be preferably RNA-free and RNase-free water.

[0067] In this application, "main solvent" means that it accounts for at least 80% of the volume of the solvent, but it can also be other proportions such as at least 90%, at least 95%, at least 98%, at least 99%, 100%, etc. In some embodiments, the solvent in aqueous solution I is water, in which case the volume percentage of water in the solvent is 100%.

[0068] In the liquefaction combination product for viscous biological samples provided in this application, after sample liquefaction is completed, the addition of a second component can neutralize the strong acidity of the liquefaction combination product and thereby adjust the pH of the system to avoid interfering with the subsequent nucleic acid detection results.

[0069] In some embodiments, the second component is a strong alkaline solution.

[0070] In this application, unless otherwise specified, a strong base aqueous solution is an aqueous solution formed by dissolving a strong base in water.

[0071] In some embodiments, the strong base mentioned above includes one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, and choline.

[0072] In some embodiments, the aforementioned viscous biological sample includes one or more of sputum, cervical mucus, nasopharyngeal swabs, and oral swabs.

[0073] Unless otherwise stated, the viscous biological samples in this application contain nucleic acids.

[0074] In some implementations, the nucleic acids in the sticky biological sample include one or more of DNA and RNA.

[0075] In some implementations, the viscous biological sample contains pathogens with nucleic acid components.

[0076] In some embodiments, the pathogen may be selected from viruses, bacteria, fungi, parasites and their eggs, or from substances that cause disease, such as tumor cells and exosomes.

[0077] In some embodiments, the bacteria may be selected from one or more of the first group and the second group; the first group includes one or more of Staphylococcus spp., Streptococcus spp., Listeria spp., Erysipelothrix spp., Nephrobacter spp., Bacillus spp., Clostridium spp., Mycobacterium spp., Actinomyces spp., Nocardia spp., Corynebacterium spp., and Rhodococcus spp.; the second group includes one or more of Bacillus anthracis, Erysipelothrix rhusiopathiae, Clostridium tetani, Listeria spp., Bacillus emphysematous, Mycobacterium tuberculosis, Escherichia coli, Proteus spp., Shigella dysenteriae, Klebsiella pneumoniae, Brucella spp., Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter spp., Yersinia spp., Legionella pneumophila, Bordetella pertussis, Bordetella parapertussis, Shigella spp., Pasteurella spp., Vibrio cholerae, and parahaemolyticus.

[0078] Another embodiment of this application provides the use of liquefied combination products in the preparation of nucleic acid detection kits.

[0079] In some embodiments of this application, the above-described applications are for non-diagnostic and non-therapeutic purposes.

[0080] In some embodiments of this application, the above-described applications are for diagnostic and therapeutic purposes.

[0081] Another embodiment of this application provides a nucleic acid detection kit, including the above-described liquefied combination product.

[0082] In some embodiments, the above-mentioned nucleic acid detection kit further includes nucleic acid detection reagents, which include one or more of primers, probes, PCR buffers, DNA polymerase, UDG enzyme, and dNTPs.

[0083] One embodiment of this application provides a pretreatment method for viscous biological samples, comprising: liquefying the viscous biological sample using a liquefaction combination product of the viscous biological sample to prepare a liquefied treatment product.

[0084] In some embodiments, the above-described step of liquefying the viscous biological sample includes steps a to c.

[0085] Step a: Mix the above-mentioned viscous biological sample with the first component and rigid particles in the above-mentioned liquefied combination product to prepare a first mixture.

[0086] Step b: Mix the first mixture with a strong alkaline solution, adjust the pH of the system to 8-10, and prepare the second mixture.

[0087] In some embodiments, the concentration of the strong base in the second mixture is 50 mmol / L to 200 mmol / L. For example, the concentration may be 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, or 200 mmol / L.

[0088] In one specific example, the concentration of the strong base in the second mixture described above is 100 mmol / L.

[0089] Step c: The second mixture above is subjected to ultrasonic treatment to prepare a liquefied product.

[0090] In some embodiments, the rigid particles mentioned above include one or more of zirconium oxide beads, zirconium silicate, glass beads, and stainless steel beads.

[0091] Understandably, the rigid particles mentioned above can accelerate the mixing process.

[0092] The material of rigid particles can be any hard material. The term "rigid" means that they do not usually break down significantly when mixing / assisting in mixing viscous biological samples, and do not release components that interfere with subsequent processes (such as at least one of the processes of nucleic acid preservation, release, enrichment, amplification, and detection).

[0093] Rigid microparticles can be used to assist in mixing viscous biological samples and the aforementioned viscous biological sample liquefaction composition, reducing processing time and increasing processing efficiency.

[0094] In some embodiments, the particle size of the rigid particles is 2 mm to 4 mm.

[0095] In some embodiments, the amount of the rigid particles used is 5 to 20.

[0096] Understandably, homogenization / grinding is a fundamental and important physical liquefaction technique in sputum processing. It uses mechanical force to forcibly disrupt the physical structure of sputum, transforming it from a viscous, heterogeneous state into a relatively homogeneous, aspirable suspension. The shearing, compressing, and impact forces generated by a mortar / pestle or a homogenizer with grinding beads directly disrupt key structures in the sputum, such as mucin polymer networks, cell clumps / aggregates, and mucus plugs / solid particles. During this disruption, encapsulated pathogens (bacteria, fungi, tuberculosis bacilli) and cells are released, improving the detection rate of subsequent tests.

[0097] In some embodiments, the frequency of the ultrasonic treatment is 10kHz to 80kHz and the duration is 60s to 200s.

[0098] As can be understood, ultrasonic liquefaction is a method that uses the power of ultrasonic vibrations to induce the formation of tiny bubbles and eddies in a liquid, thereby improving its fluid properties. Ultrasonic liquefaction can liquefy sputum in a relatively short time, improving processing efficiency. It is well-suited for different types of sputum samples, including highly viscous sputum. By adjusting the frequency and duration of the ultrasound waves, the liquefaction process can be controlled.

[0099] The liquefaction combination products for viscous biological samples described above can liquefy viscous biological samples simply, quickly, and efficiently, and are compatible with lyophilized direct amplification systems while synergistically resisting amplification inhibitors in the whole sample.

[0100] Another embodiment of this application provides a method for detecting nucleic acids in viscous biological samples for non-diagnostic and non-therapeutic purposes, including steps S100 to S300.

[0101] Step S100: Provide a viscous biological sample.

[0102] Step S200: Use the above-mentioned liquefaction combination product to liquefy the above-mentioned viscous biological sample to obtain the liquefied product.

[0103] Step S300: Use nucleic acid detection reagents to detect the target nucleic acid in the above liquefied material.

[0104] The above-mentioned method for detecting nucleic acids in viscous biological samples uses the liquefaction combination product for viscous biological samples of this application. It has the advantages of short liquefaction time, good liquefaction effect, simple operation, high repeatability and no interference with nucleic acid detection. It is suitable for processing sputum samples of different viscous consistency and has broad application prospects.

[0105] To make the objectives, technical solutions, and advantages of this application clearer and more concise, the following specific embodiments are used for illustration, but this application is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of this application and can be used to describe this application, but should not be construed as limiting the scope of this application. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0106] The present application will be further described below with reference to specific embodiments and comparative examples, but should not be construed as limiting the scope of protection of the present application.

[0107] Example 1

[0108] 1. Verification of synergistic effect of liquefying agent components

[0109] 1.1 To better verify the synergistic effect of the liquefying agent components in this application on sputum liquefaction, the following experiment was designed.

[0110] Two clinical sputum samples were selected and divided into five parallel portions, designated as samples 1 and 2. The samples were grade II and III sputum samples, respectively. The criteria for classifying the consistency of the sputum samples are as follows.

[0111] Grade I sputum is relatively rice-water-like or foamy, and leaves no residue when it comes into contact with the glass tube after direct suctioning.

[0112] Grade II sputum is slightly thicker than Grade I sputum. After suctioning, a small amount of sputum will stick to the glass tube, but it can be easily rinsed off with water.

[0113] Grade III sputum is more viscous than Grade II sputum and may be yellow or green, with a darker color. After suctioning, a large amount of sputum will remain on the glass tube wall, which is not easily washed away with water.

[0114] The clinical significance of sputum sample viscosity grade is that as viscosity increases, it indicates a more severe infection, especially grade III sputum, which is the most severe infection and relatively viscous.

[0115] Experimental Group 1: The final concentrations of the first component mixture were 50 mol / L N-acetyl-L-cysteine, 20 mmol / L guaiacol ether, and 10 mmol / L 2-N-(morphorline) ethanesulfonic acid (MES). The solvent used to prepare the reagents was ultrapure water. The second component reagent was sodium hydroxide solution with a concentration of 10 mol / L.

[0116] Experimental Group 2: 20 mmol / L guaiacol and 10 mmol / L 2-N-(morphorline)ethanesulfonic acid (MES), with ultrapure water as the solvent. 50 mol / L N-acetyl-L-cysteine ​​was omitted, and the types and concentrations of the remaining components were the same as those in the first component mixture of Experimental Group 1.

[0117] Experimental Group 3: 50 mol / L N-acetyl-L-cysteine, 20 mmol / L guaiacol ether, with ultrapure water as the solvent. 2-N-(morphorline)ethanesulfonic acid (MES) was omitted. The types and concentrations of the remaining components were the same as those in the first component mixture of Experimental Group 1.

[0118] Experimental Group 4: 50 mol / L N-acetyl-L-cysteine, 10 mmol / L 2-N-(morphorline)ethanesulfonic acid (MES), with ultrapure water as the solvent. Guaifenesin was omitted, and the types and concentrations of the remaining components were the same as those in the first component mixture of Experimental Group 1.

[0119] Experimental group 5: The composition is different from that of experimental group 1, specifically 0.4% dithiothreitol.

[0120] Treatment method for experimental groups 1-5: Add twice the volume of the first component mixture of each experimental group to the sputum sample, mix thoroughly, and add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample. Vortex intermittently and observe the liquefaction effect at 5 min and 10 min time points.

[0121] Analysis of Experimental Results: The liquefaction effects of each experimental group were compared. The comparative indicators included the time to complete liquefaction and the presence of visible sputum or excessive mucus 10 minutes after liquefaction, allowing for pipetting (a 10μL pipette was used in this example). A value of "no" for all indicators indicated complete liquefaction and good liquefaction effect. The results can be found in Table 1.

[0122] Table 1

[0123]

[0124] The results showed that omitting 2-N-(morphorline)ethanesulfonic acid (MES) from the liquefaction agent in the experimental groups had little effect on the liquefaction time of sputum (Liquefaction agent 2). This suggests that the role of 2-N-(morphorline)ethanesulfonic acid (MES) in the liquefaction agent may mainly be to buffer the pH of the system and stabilize nucleic acids, which is beneficial for subsequent nucleic acid detection. Omitting N-acetyl-L-cysteine ​​(Liquefaction agent 1 in experimental groups) significantly increased the liquefaction time, indicating that N-acetyl-L-cysteine ​​plays a key role in accelerating sputum liquefaction, especially for thick sputum of grades II and III. Omitting guaiacol (Liquefaction agent 3 in experimental groups) also increased the liquefaction time, but not as significantly as in experimental group 1, indicating that guaiacol also plays a certain synergistic role in sputum liquefaction.

[0125] 1.2 Investigation of the types and concentrations of each component in the liquefied component (first component)

[0126] The liquefaction agent formulations for each experimental group are shown in Table 2.

[0127] Experimental Group 1: The composition is the same as the first component mixture in section 1.1 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in section 1.1 of Example 1.

[0128] The liquefying agents in experimental groups 2 to 9 replaced the first component mixture in experimental group 1, and the operation steps, operation parameters, and liquefaction effect testing methods were the same as those in experimental group 1 in this example.

[0129] Treatment methods for each experimental group: Add twice the volume of the first component of the liquefaction agent of each experimental group to the sputum sample, mix thoroughly, and add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample. Intermittently shake and vortex, and observe the liquefaction effect at time points such as 5 min and 10 min.

[0130] Analysis of Experimental Results: The liquefaction effects of each experimental group were compared. The comparative indicators included the time to complete liquefaction and the presence of visible sputum or excessive mucus 10 minutes after liquefaction, allowing for pipetting (a 10μL pipette was used in this example). A value of "no" for all indicators indicated complete liquefaction and good liquefaction effect. The results can be found in Table 3.

[0131] Table 2

[0132]

[0133] Table 3

[0134]

[0135] The results showed that N-acetyl-L-cysteine ​​and guaiacol ether played key liquefaction roles in the liquefaction process of the experimental group's liquefying agent. As the concentration of these two components decreased, the liquefaction effect of the liquefying agent significantly weakened. Changes in the concentration of MES had little effect on the liquefaction effect of the liquefying agent.

[0136] 1.3 Verification of the liquefaction effect of ultrasonic function in liquefaction methods

[0137] To better examine the degree of enhancement of the liquefaction effect of the ultrasonic function in the liquefaction method of this application, the following comparative experiment was designed.

[0138] Experimental Group 1: The composition is the same as the first component mixture of Experimental Group 1 in Section 1.1 of Example 1. The operation steps, operation parameters, and liquefaction effect test methods are the same as those of Experimental Group 1 in Section 1.1 of Example 1. Add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, and intermittently vortex the sample. Observe the liquefaction effect at time points such as 2 min and 5 min.

[0139] Experimental Group 2: The composition was the same as the first component mixture of Experimental Group 1 in Section 1.1 of Example 1. The operation steps, operating parameters, and liquefaction effect testing methods were the same as those of Experimental Group 1 in Section 1.1 of Example 1. 10-20 zirconium oxide beads with a particle size of 3 mm were added according to the viscosity of the sample, and the mixture was intermittently vortexed. It was then transferred to an EP tube for ultrasonic treatment at a frequency of 10 kHz to 80 kHz for 120 seconds. The liquefaction effect was observed at time points of 2 min and 5 min.

[0140] Experimental Group 3: The composition is the same as the first component mixture of Experimental Group 2 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. Add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, and vortex intermittently. Observe the liquefaction effect at time points such as 2 min and 5 min.

[0141] Experimental Group 4: The composition is the same as the first component mixture of Experimental Group 2 in Section 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect testing methods are the same as those of Experimental Group 1 in Section 1.1 of Example 1. 10-20 zirconium oxide beads with a particle size of 3 mm are added according to the viscosity of the sample, and the mixture is intermittently vortexed. It is then transferred to an EP tube for ultrasonic treatment at a frequency of 10kHz-80kHz for 120 seconds. The liquefaction effect is observed at time points such as 2 min and 5 min.

[0142] Experimental Group 5: The composition is the same as the first component mixture of Experimental Group 5 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. Add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, and vortex intermittently. Observe the liquefaction effect at time points such as 2 min and 5 min.

[0143] Experimental Group 6: The composition is the same as the first component mixture of Experimental Group 5 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect testing methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. 10-20 zirconium oxide beads with a particle size of 3 mm are added according to the viscosity of the sample, and the mixture is intermittently vortexed. It is then transferred to an EP tube for ultrasonic treatment at a frequency of 10 kHz to 80 kHz for 120 seconds. The liquefaction effect is observed at time points such as 2 min and 5 min.

[0144] Experimental Group 7: The composition is the same as the first component mixture of Experimental Group 6 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. Add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, and vortex intermittently. Observe the liquefaction effect at time points such as 2 min and 5 min.

[0145] Experimental Group 8: The composition was the same as the first component mixture of Experimental Group 6 in Section 1.2 of Example 1. The operation steps, operating parameters, and liquefaction effect testing methods were the same as those of Experimental Group 1 in Section 1.1 of Example 1. 10-20 zirconium oxide beads with a particle size of 3 mm were added according to the viscosity of the sample, and the mixture was intermittently vortexed. It was then transferred to an EP tube for ultrasonic treatment at a frequency of 10 kHz to 80 kHz for 120 seconds. The liquefaction effect was observed at time points of 2 min and 5 min.

[0146] Experimental Group 9: The composition is the same as the first component mixture of Experimental Group 8 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. Add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, and vortex intermittently. Observe the liquefaction effect at time points such as 2 min and 5 min.

[0147] Experimental Group 10: The composition is the same as the first component mixture of Experimental Group 8 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect testing methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. 10-20 zirconium oxide beads with a particle size of 3 mm are added according to the viscosity of the sample, and the mixture is intermittently vortexed. Then it is transferred to an EP tube for ultrasonic treatment. The ultrasonic frequency is 10kHz-80kHz, and the ultrasonic treatment lasts for 120s. The liquefaction effect is observed at time points such as 2 min and 5 min.

[0148] Experimental Group 11: The composition is the same as the first component mixture of Experimental Group 9 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. Add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, and vortex intermittently. Observe the liquefaction effect at time points such as 2 min and 5 min.

[0149] Experimental Group 12: The composition is the same as the first component mixture of Experimental Group 9 in Part 1.2 of Example 1. The operation steps, operation parameters, and liquefaction effect testing methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1. 10-20 zirconium oxide beads with a particle size of 3 mm are added according to the viscosity of the sample, and the mixture is intermittently vortexed. It is then transferred to an EP tube for ultrasonic treatment at a frequency of 10 kHz to 80 kHz for 120 seconds. The liquefaction effect is observed at time points such as 2 min and 5 min.

[0150] Experimental Results Analysis: The liquefaction effects of each experimental group were compared. The comparative indicators included the time to complete liquefaction and the presence of visible sputum or excessive mucus 10 minutes after liquefaction, allowing for pipetting (a 10μL pipette was used in this example). A value of "no" for all indicators indicated complete liquefaction and good liquefaction effect. The results can be found in Table 4.

[0151] Table 4

[0152]

[0153] The results showed that the liquefaction effect was better in all experimental groups after adding the ultrasound step, indicating that the ultrasound process plays a key role in sample liquefaction, which can quickly break up small lumps of sputum, making the liquefied sample clear, and shortening the liquefaction time to 2 min to 5 min.

[0154] Example 2: Validation of the experimental effect of liquefied nucleic acid detection

[0155] To verify the effectiveness of the liquefaction method provided in this application in nucleic acid amplification after processing sputum samples, the following comparative experiment was designed.

[0156] Experimental Group 1: The composition is the same as the first component mixture in section 1.1 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in section 1.1 of Example 1.

[0157] Experimental Group 2: The composition is the same as the first component mixture of Experimental Group 2 in Part 1.2 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1.

[0158] Experimental Group 3: The composition is the same as the first component mixture of Experimental Group 5 in Part 1.2 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1.

[0159] Experimental Group 4: The composition is the same as the first component mixture of Experimental Group 6 in Part 1.2 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1.

[0160] Experimental Group 5: The composition is the same as the first component mixture of Experimental Group 8 in Part 1.2 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1.

[0161] Experimental Group 6: The composition is the same as the first component mixture of Experimental Group 9 in Part 1.2 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in Part 1.1 of Example 1.

[0162] Processing methods for each experimental group: For two clinical samples that were divided into 6 parallel groups, add 2 times the volume of the first component of the liquefaction agent of each experimental group to the sputum sample, mix thoroughly, add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample, vortex intermittently, and after complete liquefaction, transfer to EP tubes or droppers, add 10 mol / L sodium hydroxide to adjust the pH value to the appropriate pH value of 8.5 for PCR amplification, and then perform sonication at a frequency of 50 kHz for 120 seconds. After sonication, let stand for 3 minutes to allow the sample to cool before use.

[0163] The reagent used for the test was the Seven Respiratory Pathogen Nucleic Acid Detection Kit (Fluorescent PCR Method) from Sansure Biotech Inc. This reagent is a 25μL lyophilized reaction system. You can directly add 25μL of the pretreated sample to the lyophilized bulb for complete reconstitution, and then perform fluorescence quantitative PCR amplification and verification after shaking and centrifugation. The operation is simple.

[0164] Control group 1: The components and final concentrations of the liquefaction agent were: 800 mmol / L sodium hydroxide, 150 mmol / L acetylcysteine, and 5 mmol / L 2-(N-morpholino)ethanesulfonic acid. The specific steps for sputum liquefaction, lyophilization, direct amplification, and nucleic acid detection were as follows: The liquefaction agent was mixed with the sample at twice the sample volume, incubated at room temperature for 10 minutes, and then vortexed. 25 μL of the liquefied sample was then transferred to a lyophilized pellet of a seven-item respiratory pathogen nucleic acid detection kit (fluorescent PCR method), centrifuged, and then subjected to quantitative fluorescent PCR amplification for verification.

[0165] Control Group 2: The composition of the liquefaction solution was the same as that of Control Group 1, with the addition of zirconium beads from the experimental group and ultrasonic treatment. The specific steps for sputum liquefaction, lyophilization, direct amplification, and nucleic acid detection were as follows: The sample was mixed with twice the sample volume of liquefaction solution. Depending on the sample viscosity, 10-20 zirconium oxide beads with a particle size of 3 mm were added. The mixture was vortexed for 10 minutes, followed by ultrasonic treatment at a frequency of 50 kHz for 120 seconds. The mixture was then allowed to stand for 3 minutes until the sample cooled before use. 25 μL of the pretreated sample was transferred to lyophilized pellets of a seven-item respiratory pathogen nucleic acid detection kit (fluorescent PCR method), centrifuged, and then subjected to quantitative real-time PCR amplification for verification.

[0166] The experimental results are shown in Table 5 and Figure 1 , Figure 2 , Figure 3 As shown.

[0167] Table 5

[0168]

[0169] Wherein: " / " indicates that no signal value was detected and the result is negative.

[0170] The results showed that the liquefaction agent in the experimental groups exhibited a significant advantage in nucleic acid detection. Increasing the N-acetyl-L-cysteine ​​concentration in the liquefaction agent (Experimental Group 2 in Example 2) delayed the Ct value of nucleic acid detection by approximately 12 Ct, or completely inhibited the PCR reaction, resulting in no detection results. This indicates that excessively high N-acetyl-L-cysteine ​​strongly chelates magnesium ions, which are crucial in the PCR reaction, leading to a significant decrease or complete inhibition of PCR amplification efficiency. Increasing the guaiacol concentration in the liquefaction agent (Experimental Groups 3 and 4 in Example 2) delayed the Ct value of nucleic acid detection by 3-10 Ct. This may be because high concentrations of guaiacol interfere with the enzyme's active site or non-specifically bind to the DNA template or primers, hindering primer annealing or Taq enzyme extension, thus reducing PCR amplification efficiency. 2-N-(morphorline)ethanesulfonic acid (MES) is a commonly used biological buffer. Its main function is to maintain the optimal pH range of the reaction system and maintain the stability of nucleic acids. Too high or too low a pH is not conducive to PCR amplification (experimental groups 5 and 6 in Example 2).

[0171] Control groups 1 and 2 mainly used strong alkali for deliquescing, while the technical solution in Example 2 of this application mainly uses strong acid for deliquescing. Rigid particles are used to break the sample, and a strong alkali is added to neutralize the pH value while sonicating. This ensures rapid sample liquefaction while minimizing the amount of strong alkali and cations (such as sodium ions) in the sample liquefaction system, so that they do not inhibit the PCR amplification reaction in the subsequent lyophilization reagent amplification of nucleic acids.

[0172] Example 3: Comparison with Traditional Liquefaction Methods

[0173] Experimental Group 1: The composition is the same as the first component mixture in section 1.1 of Example 1, and the operation steps, operation parameters and liquefaction effect test methods are the same as those of Experimental Group 1 in section 1.1 of Example 1.

[0174] Control group 1: 1 mol / L sodium hydroxide, the solvent used to prepare the reagent was ultrapure water.

[0175] Control group 2: 0.9% (w / v) physiological saline, with ultrapure water as the solvent used to prepare the reagents.

[0176] Control group 3: 2% (w / v) dithiothreitol solution (DTT), the solvent used to prepare the reagent was ultrapure water.

[0177] Control group 4: Commercial preservation solution (purchased from Shanghai Aiyan Biotechnology Co., Ltd.; product number: BSC83S1).

[0178] Viscous biological samples: Twelve clinical sputum samples were selected and divided into five parallel aliquots, designated as samples 1 to 12. The samples contained samples of different viscosities: samples 1 and 2 were grade I sputum samples, samples 3 to 7 were grade II sputum samples, and samples 8 to 12 were grade III sputum samples.

[0179] Liquefaction method for the experimental group: Add 1-3 times the volume of sputum liquefaction solution to the sputum sample and mix well. At the same time, add 10-20 zirconium oxide beads with a particle size of 3 mm according to the viscosity of the sample. Vortex intermittently. After complete liquefaction, proceed to the next step (about 2 min-5 min). Mix the liquefied sample with 10 mol / L sodium hydroxide at a ratio of 198:2 and transfer it to an EP tube to prepare a second mixture. The concentration of sodium hydroxide in the second mixture is 100 mmol / L. Then, the second mixture is sonicated at a frequency of 50 kHz for 120 s.

[0180] Control group experimental method: Add twice the volume of sputum liquefaction agent 1-4 to the sputum sample, mix thoroughly, and vortex intermittently. Observe the liquefaction effect at different time points within 30 minutes.

[0181] Analysis of experimental results: The liquefaction effects of each experimental group and each control group were compared. The comparison indicators included the time to complete liquefaction, whether there was visible sputum or a large amount of mucus after 5 minutes of liquefaction, and whether pipetting could be performed (a 10μL pipette was used in this example). "No" in all indicators indicated complete liquefaction and good liquefaction effect. The results can be found in Table 6.

[0182] Table 6

[0183]

[0184]

[0185] “ / ” indicates that no liquefaction was observed within 30 minutes.

[0186] The results showed that, compared with the traditional chemical dissolution method, the liquefaction method of this application exhibited shorter liquefaction time and better liquefaction effect for sputum samples of different viscosities, significantly improving the experimental results. In particular, for grade II and grade III sputum samples, the experimental group liquefaction method could completely liquefy the sputum within 2 min to 5 min, while the control group solution could not completely liquefy the sputum even after 30 min.

[0187] Based on the above experimental data, the liquefaction method of this application, through the optimized combination of N-acetyl-L-cysteine, guaiacol ether, and 2-N-(morphorline)ethanesulfonic acid (MES) in the liquefying agent, and through physical methods such as adding zirconium oxide beads and ultrasound, demonstrates significant advantages in sputum liquefaction, lyophilized direct amplification, and nucleic acid detection. It solves the technical problems existing in traditional methods and provides an efficient and reliable solution for the rapid and effective processing of sputum samples.

[0188] 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.

[0189] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A liquefaction combination product for viscous biological samples, characterized in that, Includes the first component and the second component; The first component comprises: 10 mmol / L to 100 mmol / L acetylcysteine, 5 mmol / L to 50 mmol / L guaiacol, and 2 mmol / L to 20 mmol / L 2-morpholinoethanesulfonic acid; The second component includes a strong alkali solution, wherein the concentration of the strong alkali in the strong alkali solution is 5 mol / L to 20 mol / L.

2. The liquefaction combination product for viscous biological samples according to claim 1, characterized in that, The liquefied combination product satisfies at least one of the following characteristics: (1) The concentration of acetylcysteine ​​is 50 mmol / L to 100 mmol / L; (2) The concentration of the guaiacol is 20 mmol / L to 50 mmol / L; (3) The concentration of the 2-morpholinoethanesulfonic acid is 5 mmol / L to 20 mmol / L; (4) The strong base includes one or more of sodium hydroxide and potassium hydroxide.

3. The liquefaction combination product for viscous biological samples according to any one of claims 1 to 2, characterized in that, The viscous biological sample includes one or more of the following: sputum, cervical mucus, nasopharyngeal swabs, and oral swabs.

4. The use of the liquefaction combination product of viscous biological samples according to any one of claims 1 to 3 in the preparation of a nucleic acid detection kit.

5. A nucleic acid detection kit, characterized in that, The product includes a liquefied combination of viscous biological samples as described in any one of claims 1 to 3.

6. The nucleic acid detection kit according to claim 5, characterized in that, The nucleic acid detection kit also includes nucleic acid detection reagents, which include one or more of primers, probes, PCR buffer, DNA polymerase, UDG enzyme, and dNTPs.

7. A pretreatment method for viscous biological samples, characterized in that, Includes the following steps: The viscous biological sample is liquefied using the liquefaction combination product of any one of claims 1 to 3 to prepare a liquefied treatment.

8. The pretreatment method for viscous biological samples according to claim 7, characterized in that, The step of liquefying the viscous biological sample includes the following steps: The viscous biological sample is mixed with the first component and rigid particles in the liquefied composite product to prepare a first mixture; The first mixture is mixed with a strong alkaline solution, and the pH of the system is adjusted to 8-10 to prepare a second mixture; and the second mixture is subjected to ultrasonic treatment to prepare the liquefied product.

9. The pretreatment method for viscous biological samples according to claim 8, characterized in that, The pretreatment method for the viscous biological samples meets one or more of the following conditions: (1) The rigid particles include one or more of zirconium oxide beads, zirconium silicate, glass beads, and stainless steel beads; (2) The particle size of the rigid particles is 2 mm to 4 mm; (3) The amount of the rigid particles used is 5 to 20; and, (4) The frequency of the ultrasonic treatment is 10kHz~80kHz and the time is 60s~180s.

10. A method for detecting nucleic acids in viscous biological samples for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: Provide viscous biological samples; The viscous biological sample is liquefied using the liquefaction combination product of any one of claims 1 to 3 to prepare a liquefied product; as well as, The target nucleic acid in the liquefied material is detected using nucleic acid detection reagents.

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