Liquefaction kit for viscous biological samples, nucleic acid amplification system, nucleic acid detection system, processing method and use

Abstract

The present application provides a liquefaction combination product for viscous biological samples, a nucleic acid amplification system, a nucleic acid detection system, a processing method and applications. The liquefaction combination product comprises a component a and further comprises or does not comprise a component b; the component a is an aqueous solution I or a mother liquor of the aqueous solution I comprising the following components: acetylcysteine 3% to 15% (w / v), sodium chloride 20 mM to 500 mM, potassium chloride 50 mM to 120 mM and 50 to 1000 mM strong base, calculated as monobasic base; the component b is citric acid or an aqueous solution thereof. Through the synergistic effect of the above components, the liquefaction combination product can directly and quickly fully liquefy the viscous biological sample at room temperature, can avoid the sample pollution problem caused by aerosol generated by heating, and does not need to perform complex steps such as heating. The sample processed by the liquefaction combination product can stably store nucleic acid, and can realize extraction-free amplification and extraction-free detection.

Description

Technical Field

[0001] This invention relates to the field of medical sample processing and nucleic acid extraction and detection technology, specifically to a liquefaction combination product for viscous biological samples, a nucleic acid amplification system, a nucleic acid detection system, a processing method, and its application. Background Technology

[0002] With the rapid development of technologies such as quantitative real-time PCR and multiplex PCR in the field of pathogen detection, the demand for detecting the presence of specific pathogens through sputum sample analysis is increasing. However, sputum samples are characterized by high viscosity, high protein content, and complex composition, containing mucin and other proteins (such as immunoproteins), various enzymes, exfoliated cells, microorganisms, and other inhaled impurities, making direct detection inconvenient. Clinically, sputum sample testing requires liquefaction treatment beforehand.

[0003] Common sputum liquefaction methods include the sodium hydroxide method, the DTT (dithiothreitol) method, and the protease method. The sodium hydroxide method is the most commonly used and relatively simple. It uses a high-concentration (usually at least 4 wt%) sodium hydroxide solution to liquefy sputum at 60℃–80℃ (room temperature is also acceptable). However, when used for nucleic acid detection, the highly alkaline environment can easily cause nucleic acid loss. The protease method utilizes proteases to digest sputum mucin. This method is time-consuming, requires expensive proteases, and has low mucin digestion efficiency. The DTT (dithiothreitol) method is currently the most commonly used sputum liquefaction method. It uses thiol-containing (-SH) DTT to break down mucin, the main component causing sputum viscosity, using PBS (phosphate-buffered saline) to provide physiological buffering. Ethanol is often added to fix cells. This method is time-consuming, uses expensive DTT, and DTT has poor stability, requiring low-temperature storage. Furthermore, DTT has some toxicity, making it unsuitable for large-scale clinical processing of sputum samples. Furthermore, due to the high viscosity of sputum samples, uneven mixing is easily encountered during conventional liquefaction processes. Nucleic acids, especially RNA, in sputum samples are extremely unstable and typically degrade within hours under ambient conditions. However, in clinical testing, it is often impossible to process and detect nucleic acids in sputum samples promptly. Therefore, it is essential to develop a method that can rapidly and thoroughly liquefy sputum and effectively protect the nucleic acids in the sample. Currently, there are numerous published inventions regarding sputum liquefaction and nucleic acid protection. However, these methods generally suffer from problems such as complex composition, high cost, complex operation, and incomplete sample mixing and liquefaction.

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

[0005] Therefore, the present invention aims to provide a liquefaction combination product for viscous biological samples. The liquefaction components in this product can liquefy viscous biological samples simply, rapidly, and efficiently, and the protective components can effectively preserve the liquefied samples. This liquefaction combination product can be used in nucleic acid amplification systems and nucleic acid detection systems. The aforementioned liquefaction combination product can also be used for extraction-free amplification and extraction-free detection of viscous biological samples.

[0006] In a first aspect of the invention, a liquefaction combination product of a viscous biological sample is provided, comprising component a and may or may not contain component b.

[0007] The a component is an aqueous solution I containing the following components or the mother liquor of the aqueous solution I: acetylcysteine ​​3% to 15% (w / v), sodium chloride 20mM to 500mM, potassium chloride 50mM to 120mM, and a strong base; wherein, as a monobasic base, the concentration of the strong base in the aqueous solution I is 50mM to 1000mM.

[0008] Component b is citric acid or an aqueous solution of citric acid.

[0009] In a second aspect of the invention, a nucleic acid amplification system for viscous biological samples is provided, comprising a liquefied combination product of the viscous biological samples described in the first aspect of the invention, and further comprising nucleic acid amplification reagents.

[0010] In a third aspect of the invention, a nucleic acid detection system for viscous biological samples is provided, comprising a liquefied combination product of the viscous biological samples described in the first aspect of the invention, and further comprising nucleic acid detection reagents.

[0011] In a fourth aspect of the present invention, a method for processing viscous biological samples is provided, comprising the following steps:

[0012] Aqueous solution I was used to liquefy viscous biological samples to obtain liquefied material;

[0013] The composition of the aqueous solution I is as defined in the first aspect of the present invention.

[0014] In a fifth aspect of the invention, the following is provided:

[0015] The application of the liquefaction combination product for viscous biological samples according to the first aspect of the present invention in the liquefaction of viscous biological samples, wherein the liquefaction combination product includes the a component and may or may not contain the b component; or,

[0016] The application of the liquefaction combination product for viscous biological samples according to the first aspect of the present invention in the preservation of viscous biological samples, wherein the liquefaction combination product comprises component a and component b; or,

[0017] The liquefaction combination product of viscous biological samples according to the first aspect of the present invention, or the application of the nucleic acid amplification system of viscous biological samples according to the second aspect of the present invention in the extraction-free amplification of viscous biological samples, wherein the liquefaction combination product includes the a component and may or may not contain the b component; or,

[0018] The liquefaction combination product of viscous biological samples according to the first aspect of the present invention, or the nucleic acid amplification system of viscous biological samples according to the second aspect of the present invention, or the nucleic acid detection system of viscous biological samples according to the third aspect of the present invention, is applied in the extraction-free detection of viscous biological samples, wherein the liquefaction combination product includes the a component and may or may not contain the b component.

[0019] The liquefaction combination product for viscous biological samples provided by this invention can liquefy sputum using a certain ratio and concentration of acetylcysteine ​​and a strong alkali. Sodium chloride and potassium chloride protect nucleic acids by coordinating the ion balance inside and outside the cell membrane. It can achieve simple, rapid, and efficient liquefaction of viscous biological samples at room temperature without special high-temperature heating treatment. The liquefaction time is short (e.g., ≤15 min, further ≤10 min). Further treatment of the liquefaction product with citric acid can improve the stability of nucleic acids in the sample and extend the sample preservation time. The liquefaction combination product provided by this invention is well compatible with nucleic acid extraction and amplification. In addition, the liquefaction combination product provided by this invention can also achieve extraction-free amplification and extraction-free detection.

[0020] Through the synergistic effect of the above components, viscous biological samples can be rapidly and fully liquefied at room temperature, avoiding sample contamination problems caused by aerosol generation during heating in the traditional sodium hydroxide method, and eliminating the need for complex heating procedures. Samples treated with the combined components A and B of the above liquefaction product can stably preserve nucleic acids for extended periods. Nucleic acid amplification or detection can also be performed directly without nucleic acid extraction. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the result of the stability amplification curve of a DNA pathogen culture sample in one embodiment of the present invention;

[0023] Figure 2The results of the stability amplification curve of a DNA pathogenic bacteria culture sample (No. 1) in a commercially available preservation solution;

[0024] Figure 3 This is the stability amplification curve result of DNA pathogen culture sample No. 2 in one embodiment of the present invention;

[0025] Figure 4 The results of the stability amplification curve of a DNA pathogen culture sample (No. 2) in a commercially available preservation solution;

[0026] Figure 5 This is the stability amplification curve result of DNA pathogen culture sample No. 3 in one embodiment of the present invention;

[0027] Figure 6 The results of the stability amplification curve of a DNA pathogen culture sample (No. 3) in a commercially available preservation solution;

[0028] Figure 7 This is the stability amplification curve result of RNA virus culture sample No. 1 in one embodiment of the present invention;

[0029] Figure 8 The results of the stability amplification curve of RNA virus culture sample (No. 1) in a commercial preservation solution;

[0030] Figure 9 This is the stability amplification curve result of RNA virus culture sample No. 2 in one embodiment of the present invention;

[0031] Figure 10 The results of the stability amplification curve of RNA virus culture sample (2) in a commercial preservation solution;

[0032] Figure 11 This is the matrix influence test amplification curve result for sample No. 1 (black curve represents the present invention) in one embodiment of the present invention;

[0033] Figure 12 The matrix influence test amplification curve results for sample No. 2 (black curve represents the present invention) in one embodiment of the present invention. Detailed Implementation

[0034] The present invention will now be described in further detail 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 invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention 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 invention, and the equivalent forms obtained also fall within the protection scope of the present invention. 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 specific details are set forth in the following description to provide a fuller understanding of the invention. It should be understood that the present invention can be implemented without one or more of these details.

[0035] 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 invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0036] the term

[0037] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0038] In this invention, terms such as "multiple," "various," and "multiple times" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0039] In this invention, unless otherwise stated, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances of "one or more" are also understood in the same way unless otherwise stated.

[0040] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0041] In this document, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0042] In this document, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments and should be understood not to limit the scope of protection of this invention. If multiple "preferred" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferred" term shall be independent.

[0043] In this invention, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this invention. In this invention, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0044] In this invention, 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 the quantity.

[0045] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

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

[0047] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the distribution of selectable values ​​within a numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0048] In this article, unless otherwise specified, "approximately" means within a certain range above and below the given number, and the range of fluctuation may vary depending on the type and value of the given number. For example, a range of ±10%, ±5%, ±2%, ±1% is allowed.

[0049] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0050] In this invention, the term "room temperature" or "normal temperature" generally refers to 4℃ to 35℃, for example, 20℃ ± 5℃. In some embodiments of this invention, "room temperature" or "normal temperature" refers to 10℃ to 30℃. In some embodiments of this invention, "room temperature" or "normal temperature" refers to 18℃ to 35℃. In some embodiments of this invention, "room temperature" or "normal temperature" refers to 20℃ to 30℃.

[0051] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0052] In this invention, if the unit of a data range is only followed by the right endpoint, it indicates that the units of the left and right endpoints are the same. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0053] In this invention, w / v represents the mass-to-volume ratio, and % (w / v) represents the mass percentage contained in a certain volume of the mixture. For example, a concentration of 5% (w / v) of substance A in the mixture means that 5 grams of substance A are contained in every 100 milliliters of the mixture.

[0054] In this invention, wt% represents the mass percentage concentration, referring to the percentage by mass contained in a certain mass mixture. For example, a concentration of 5wt% for substance A in a mixture means that 5 grams of substance A are contained in every 100 grams of the mixture.

[0055] In this invention, "within N1 minutes" means ≤ N1 minutes. Taking "within 15 minutes" as an example, unless otherwise specified, it refers to ≤ 15 minutes.

[0056] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0057] The mass or weight of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship of mass or weight between the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the mass or weight mentioned in the embodiments of this invention can be units known in the chemical industry, such as μg, mg, g, and kg.

[0058] In this invention, where the method involves multiple steps, unless otherwise explicitly stated herein, the execution of these steps is not strictly limited in order and can be performed in any order other than that 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.

[0059] In a first aspect, the present invention provides a liquefaction combination product for viscous biological samples, comprising component a, and may or may not contain component b; wherein component a is a liquefaction-compatible component containing acetylcysteine, sodium chloride, potassium chloride, and a strong base, and component b is a sample preservation-compatible component containing citric acid. The liquefaction combination product for viscous biological samples provided by the present invention includes at least component a, and may or may not contain component b. Using an aqueous solution I prepared from component a as a liquefying agent to liquefy viscous biological samples allows for simple, rapid, and efficient liquefaction of viscous biological samples at room temperature; after reducing the sample viscosity, component b can be used to adjust the pH of the system to a suitable range. The combined use of components a and b enables long-term preservation of viscous biological samples liquefied from aqueous solution I, effectively inhibiting nucleic acid degradation, ready for subsequent release-free and extraction-free amplification or detection.

[0060] Samples processed by this liquefaction combination product can stably preserve nucleic acids and can also achieve extraction-free amplification and extraction-free detection.

[0061] In some embodiments, a liquefaction combination product of a viscous biological sample is provided, which includes component a and may or may not contain component b;

[0062] The a component is an aqueous solution I containing the following components or the mother liquor of the aqueous solution I: acetylcysteine ​​3% to 15% (w / v), sodium chloride 20mM to 500mM, potassium chloride 50mM to 120mM, and a strong base; wherein, as a monobasic base, the concentration of the strong base in the aqueous solution I is 50mM to 1000mM.

[0063] Component b is citric acid or an aqueous solution of citric acid.

[0064] The liquefaction combination product for viscous biological samples provided by this invention can liquefy viscous biological samples by using a certain ratio and concentration of acetylcysteine ​​and a strong base. Sodium chloride and potassium chloride protect nucleic acids by coordinating the ion balance inside and outside the cell membrane. It can achieve simple, rapid and efficient liquefaction of viscous biological samples at room temperature without special high-temperature heating treatment. The liquefaction time is short (e.g., ≤15 min, further ≤10 min). Further treatment of the liquefaction product with citric acid can improve the stability of nucleic acids in the sample and extend the sample preservation time. The liquefaction combination product provided by this invention is well compatible with nucleic acid extraction and amplification. In addition, the liquefaction combination product provided by this invention can also achieve extraction-free amplification and extraction-free detection.

[0065] Through the synergistic effect of the above components, viscous biological samples can be rapidly and fully liquefied at room temperature, avoiding sample contamination problems caused by aerosol generation during heating in the traditional sodium hydroxide method, and eliminating the need for complex heating procedures. Samples treated with the combined components A and B of the above liquefaction product can stably preserve nucleic acids for extended periods. Nucleic acid amplification or detection can also be performed directly without nucleic acid extraction.

[0066] In this invention, components a and b should, in principle, be packaged separately. Components a can be packaged individually or in combination in one or more containers, and then mixed before use. In some preferred embodiments, components a are packaged as a mixture in one container. Components b can be packaged individually or in combination in one container.

[0067] In this invention, the terms "biological sample," "sample," etc., refer to animal samples; tissues or organs, tissue lysates, or other biological samples that may be 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 thereof), 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 cellular, nuclear, or cell-free nucleic acids. In particular, the biological samples of this invention are preferably derived from bodily fluids, including liquids, semi-solids, aerated liquids, liquid-gas mixtures, etc., derived from animals. Such bodily fluids may include, but are not limited to, saliva, sputum, serum, plasma, blood, urine, mucus, sweat, tears or other eye fluids, ear fluids, face fluids (e.g., from blisters or sores), gastric juice or gastric juice, fecal fluid, pancreatic juice or juice, semen, lactation or assay products, cerebrospinal fluid, fluid bone marrow or lymph.

[0068] In this invention, "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. 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 cytoplasmic matrix surrounding secretory cells. "Sputum" refers to viscous material contained in or expelled from the nasal or oral cavity of mammals (typically expelled from the respiratory tract).

[0069] In some embodiments, the viscous biological samples targeted by this invention are sputum, cervical mucus, nasopharyngeal swabs, or oral swabs. Further, taking sputum as an example, the viscous biological sample reaches a consistency of level 3, and the classification criteria can be found in section 1.1 of Example 1 below.

[0070] In this invention, unless otherwise stated, the viscous biological samples contain nucleic acids.

[0071] In this invention, the term "nucleic acid" refers to a polymer of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and includes naturally occurring (adenosine, guanidine, cytosine, uracil, and thymidine), non-natural, and modified nucleic acids. The term is not limited by the length of the polymer (e.g., the number of monomers). Nucleic acids can be single-stranded or double-stranded and will generally contain a 5'-3' phosphodiester bond, although in some cases, nucleotide analogs may have other bonds; nucleic acids can also be modified, such as methylated or demethylated. Nucleic acids can be DNA, RNA, or a combination thereof, i.e., nucleic acids can include at least one of DNA and RNA; RNA can be mRNA, lncRNA, small ncRNA, tiny ncRNA (e.g., siRNA, miRNA, and piRNA), tRNA, rRNA, snRNA, snoRNA, or telomerase RNA. Nucleic acids can be in the form of short oligonucleotides, long oligonucleotides, or polynucleotides. Nucleic acids can also be double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA. In some specific implementations, nucleic acids can be genes, cDNA molecules, mRNA, tRNA, rRNA, non-coding RNA molecules, and fragments of the above nucleic acid forms, such as oligonucleotides.

[0072] In some implementations, the nucleic acids in the sticky biological sample include at least DNA. DNA is more stable to preserve than RNA.

[0073] In some implementations, the viscous biological sample contains pathogens with nucleic acid components.

[0074] Pathogens can be selected from viruses, bacteria, fungi, parasites and their eggs, or from substances that cause disease, such as tumor cells and exosomes.

[0075] The virus may be selected from one or more of the following families: adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, flaviviridae, hepeviridae, mononegavirales, nidovirales, picornaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, and togaviridae.

[0076] 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 the following: 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 the following: 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.

[0077] In this invention, the terms "first" and "second" in "first group" and "second group" 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" and "second" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0078] The fungi may be selected from one or more of the following: *Coccidioides pulmonarius*, *Coccidioides pulmonarius*, *Histoplasma capsulatum*, *Histoplasma dulcis*, *Blastomyces lobotomyces*, *Paracoccidioides brasiliensis*, *Blastomyces dermatitidis*, *Sporothrix schenckii*, *Penicillium marneffei*, *Candida albicans*, *Candida glabrata*, *Candida tropicalis*, *Candida lucida*, *Aspergillus*, *Candida schoenleinii*, *Spermia pulmonata*, *Spermia pulmonata*, *Spermia dermatitidis*, *Geotrichum candida*, *Polygonum borelli*, *Cryptococcus neoformans*, *Hydrocotyle oryzae*, *Rhizopus oryzae*, *Mucor amurensis*, *Pterococcus argentea*, *Candida racemosa*, *Otocinus spp.*, *Otocinus heterosporus*, *Sinospora spp.*, *Hydrocotyle spp.*, and *Hydrocotyle globosum*.

[0079] The parasites may be selected from one or more of the following: intestinal parasites, liver parasites, lung parasites, brain tissue parasites, blood vessel parasites, lymphatic vessel parasites, muscle tissue parasites, intracellular parasites, bone tissue parasites, and intraocular parasites.

[0080] Aqueous solution I is an aqueous solution containing the following components: acetylcysteine ​​3%–15% (w / v), sodium chloride 20 mM–500 mM, potassium chloride 50 mM–120 mM, and a strong base; wherein, calculated as a monobasic base, the concentration of the strong base in aqueous solution I is 50 mM–1000 mM. The component concentrations of aqueous solution I correspond to the ready-to-use concentrations, that is, aqueous solution I at this concentration can be directly mixed with viscous biological samples to achieve liquefaction treatment of viscous biological samples.

[0081] The mother liquor of aqueous solution I is a concentrated solution of aqueous solution I, and unless otherwise specified, it is based on volumetric concentration. Before use, the mother liquor of aqueous solution I is diluted by a certain volume factor using an aqueous solvent to obtain aqueous solution I. The aqueous solvent can be water or an aqueous solution that does not affect the concentration of the aforementioned components or the pH range of the system. Addition is permitted as long as the solute in the aqueous solution does not affect the liquefaction effect of component a and the subsequent amplification and detection effects. In some preferred embodiments, the aqueous solvent is water.

[0082] The volume factor by which the mother liquor of aqueous solution I is diluted to aqueous solution I can be denoted as the dilution factor D. This dilution factor D is not particularly limited, as long as it allows for reasonable control of the concentration of each component in component a. This dilution factor can be a volume dilution of 1.5 to 100 times, or a dilution of 2 to 50 times, or it can be selected from any of the following dilution factors or any range of two dilution factors: 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 times, etc.

[0083] In some embodiments, the concentration of acetylcysteine ​​in aqueous solution I is 3% to 15% (w / v), which corresponds to approximately 306.75 mM to 920.25 mM based on the molecular weight of acetylcysteine ​​of 193 Da. The concentration of acetylcysteine ​​in aqueous solution I can also be selected from any of the following concentrations or a range consisting of two of the following concentrations, expressed as a percentage by mass (w / v): 3%, 3.2%, 3.4%, 3.5%, 3.6%, 3.8%, 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.4%, 5.5%, 5.6%, 5.8%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, etc., and can also be selected from any of the following concentration ranges: 3.5%–12% (w / v), 4%–10% (w / v), etc. If the concentration of acetylcysteine ​​is too low, the liquefaction effect may be poor after mixing with viscous biological samples; if the concentration of acetylcysteine ​​is too high (e.g., 15% (w / v)), it may form a gel-like substance, which will prevent the liquefaction reagent (aqueous solution I) formed by component a from mixing further with the sample and carrying out the liquefaction action, thus also leading to a poor liquefaction effect.

[0084] In aqueous solution I, a strong base is used to provide a suitable alkaline environment for liquefying viscous biological samples, and the pH of the system can be adjusted by controlling the concentration of the strong base.

[0085] In this invention, the term "strong base" preferably refers to, but is not limited to, a substance whose anions, when ionized in aqueous solution, are entirely 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 involved in this invention can be an organic strong base or an inorganic strong base.

[0086] The strong bases involved in this invention may include, but are not limited to, one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, francium hydroxide, cesium hydroxide, calcium hydroxide, choline, silver hydroxide, thallium hydroxide, quaternary ammonium bases, strontium hydroxide, barium hydroxide, radium hydroxide, and diamminesilver hydroxide.

[0087] In some embodiments, the strong base in aqueous solution I includes one or more inorganic strong bases.

[0088] In some preferred embodiments, the strong base in aqueous solution I is an inorganic strong base. In some preferred embodiments, the strong base in aqueous solution I is a monobasic strong base. In some preferred embodiments, the strong base in aqueous solution I is sodium hydroxide, potassium hydroxide, or a combination thereof, that is, including at least one of sodium hydroxide and potassium hydroxide.

[0089] In some embodiments, the concentration of the strong base in aqueous solution I (the corresponding molar concentration is denoted as C1) is 50 mM to 1000 mM, based on a monobasic base. When mixed with a viscous biological sample in an appropriate ratio, it can provide a strongly alkaline environment of pH ≥ 10 (further, pH ≥ 11, even further, pH ≥ 12, and even further, pH 12 to 14) for the liquefaction system. As a monobasic base, the concentration of the strong base in aqueous solution I can be selected from any one of the following concentrations or a range consisting of any two of the following concentrations: 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 112.5mM, 115mM, 120mM, 140mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 700mM, 7 50mM, 800mM, 900mM, 1000mM, etc., or can be selected from any of the following concentration ranges: 100mM~1000mM, 100mM~800mM, 100mM~750mM, 100mM~600mM, 100mM~550mM, 112.5mM~1000mM, 112.5mM~800mM, 112.5mM~750mM, 112.5mM~600mM, 112.5mM~550mM, etc.

[0090] In this invention, "based on a monobasic base" refers to the hydroxide ion concentration when the strong base ionizes 100% of its hydroxide (OH) ions into corresponding hydroxide ions. If a strong base molecule can ionize k hydroxide ions, then, based on a monobasic base, the concentration of the strong base in aqueous solution I is denoted as C1 (in molar concentration), and the molar concentration of the strong base molecules to be added is C1 / k. For a monobasic strong base, the molar concentration (in molecular concentration) of the added strong base substance is numerically equal to the concentration of hydroxide ions it ionizes.

[0091] In this invention, molar concentration is involved, and 1 mM equals 1 mmol / L.

[0092] In some embodiments, the sodium chloride concentration in aqueous solution I is 20 mM to 500 mM. The sodium chloride concentration in aqueous solution I can be selected from any of the following concentrations or a range consisting of any two of the following concentrations: 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 150 mM, 160 mM, 180 mM, 200 mM, 240 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 480 mM, 500 mM, etc., or can be selected from any of the following ranges: 20 mM to 250 mM, 150 mM to 500 mM, 150 mM to 250 mM.

[0093] In some embodiments, the potassium chloride concentration in aqueous solution I is 50 mM to 120 mM. The potassium chloride concentration in aqueous solution I can be selected from any of the following concentrations or a range consisting of any two of the following concentrations: 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, etc., or can be selected from any of the following ranges: 60 mM to 120 mM, 50 mM to 100 mM, 60 mM to 100 mM.

[0094] Sodium chloride and potassium chloride protect nucleic acids by coordinating the ion balance inside and outside the cell membrane, minimizing the impact of liquefaction on nucleic acid structural stability. If the concentrations of sodium chloride and potassium chloride are too low, it may cause the cell membrane to rupture and degrade the nucleic acid; if the concentrations are too high, it may lead to poor sample homogeneity.

[0095] In the liquefaction combination product for viscous biological samples provided by this invention, after sample liquefaction is completed, the addition of component b can generate sodium citrate and neutralize the pH value, thereby adjusting the pH of the system. This can protect nucleic acids from multiple aspects and extend the preservation time of nucleic acids in the liquefied sample.

[0096] In this invention, component b is citric acid or an aqueous solution of citric acid. In use, citric acid can be added directly to the liquefied mixture. To better control the amount of citric acid added and more precisely adjust the pH of the system, it can also be added as an aqueous solution of citric acid.

[0097] In some embodiments, component b is an aqueous solution of citric acid. In this invention, unless otherwise stated, the aqueous solution of citric acid refers to an aqueous solution formed by dissolving citric acid in water.

[0098] In some embodiments, the citric acid concentration in the aqueous citric acid solution as component b is 10% to 30% (w / v). The citric acid concentration in the aqueous citric acid solution as component b can also be any of the following concentrations or a range selected from any two of the following concentrations, expressed as a percentage (% (w / v)): 10%, 12%, 15%, 16%, 18%, 20%, 24%, 25%, 26%, 28%, 30%, etc., and can also be selected from any of the following concentration ranges: 15% to 30% (w / v), 10% to 25% (w / v), 15% to 25% (w / v).

[0099] In some embodiments, solution I comprises the following components: 4%–10% (w / v) acetylcysteine, 150 mM–250 mM sodium chloride, 60 mM–100 mM potassium chloride, and 100 mM–600 mM of an inorganic strong base, wherein the concentration of the inorganic strong base is calculated as a monobasic base. In this case, the liquefaction effect is better, and a good liquefaction effect can be achieved within 5 minutes.

[0100] In this invention, unless otherwise specified, the main solvent in aqueous solution I is water. The water used in this invention can be distilled water, purified water, filtered water, deionized water, sterile water, etc. In some embodiments, the main solvent in aqueous solution I is sterile water. Unless otherwise specified, the water used in this invention is preferably nucleic acid-free and nuclease-free. In some embodiments, the water used in this invention may also be preferably RNA-free and RNase-free.

[0101] In this invention, "main solvent" means that it accounts for at least 80% of the volume of the solvent, but 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%.

[0102] The desired liquefaction effect of component a in this invention can be achieved without the addition of nuclease inhibitors (including RNase inhibitors and DNase inhibitors). However, it should be understood that adding one or more of RNase inhibitors and DNase inhibitors to component a of this invention is also within the scope of protection of this invention. The aforementioned RNase inhibitors and DNase inhibitors are known to those skilled in the art. For example, the RNase inhibitor may be, but is not limited to, diethyl pyrocarbonate. For example, the DNase inhibitor may be, but is not limited to, ethylenediaminetetraacetic acid.

[0103] In a second aspect of the invention, a nucleic acid amplification system for viscous biological samples is provided, comprising a liquefied combination product of the viscous biological sample described in the first aspect of the invention, and further comprising nucleic acid amplification reagents. The definition of nucleic acid is the same as described above.

[0104] The term "nucleic acid amplification reagent" refers to reagents used to amplify nucleic acids.

[0105] When the term "amplifying" or "amplification" appears in the context of the term "nucleic acid," it refers to the production of multiple copies of a polynucleotide, or a portion of a polynucleotide, typically starting from a small number of polynucleotides (e.g., as few as a single polynucleotide molecule), where the amplification product or amplicon is usually detectable. Polynucleotide amplification includes a variety of chemical and enzymatic methods. Amplification is the production of multiple copies of DNA from one or more copies of a target or template DNA molecule during polymerase chain reaction (PCR), rolling circle amplification (RCA), or ligase chain reaction (LCR). Amplification is not limited to the strict replication of the starting molecule. For example, producing multiple cDNA molecules from a limited amount of RNA in a sample using reverse transcription RT-PCR is a form of amplification. Furthermore, producing multiple RNA molecules from a single DNA molecule during transcription is also a form of amplification. Amplification can be used for library preparation prior to sequencing.

[0106] In a third aspect of the invention, a nucleic acid detection system for viscous biological samples is provided, comprising a liquefied combination product of the viscous biological sample described in the first aspect of the invention, and further comprising nucleic acid detection reagents. The definition of nucleic acid is consistent with that described above.

[0107] The term "nucleic acid test reagent" refers to reagents used to detect nucleic acids.

[0108] The methods and reagents for detecting nucleic acids are well known to those skilled in the art.

[0109] Nucleic acid detection methods can rely on or not rely on nucleic acid amplification. As used herein, the term "nucleic acid detection" refers to any method that determines the nucleotide composition of a target nucleic acid, and can be quantitative or qualitative.Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays, (Hologic, Inc.) and described in, for example, U.S. Patent Nos. US5,846,717, US5,985,557, US5,994,069, US6,001,567, US6,090,543 and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999); Hall et al., PNAS, USA, 97:8272 (2000); and US2009 / 0253142, each of which is incorporated herein by reference in its entirety for all purposes); enzyme mismatch cleavage methods ( For example, Variagenics (US Patent Nos. US6,110,684, US5,958,692, US5,851,770, the entire contents of which are incorporated herein by reference); polymerase chain reaction (PCR) as described above; branching hybridization methods (e.g., Chiron, US Patent Nos. US5,849,481, US5,710,264, US5,124,246, and US5,624,802, the entire contents of which are incorporated herein by reference); rolling circle replication (e.g., US Patent Nos. US6,210,884, US6,183,960, and US6,235,502, the entire contents of which are incorporated herein by reference); nucleic acid sequence-dependent amplification detection techniques, Nuclearacid Sequence-based amplification, NASBA (e.g., U.S. Patent No. US5,409,818, the entire contents of which are incorporated herein by reference); molecular beacon technology (e.g., U.S. Patent No. US6,150,097, the entire contents of which are incorporated herein by reference); E-sensor technology (Motorola, U.S. Patent Nos. US6,248,229, US6,221,583, US6,013,170, and US6,063,573, the entire contents of which are incorporated herein by reference); circular probe technology (e.g., U.S. Patent Nos. US5,403,711, US5,011,769, and US5,660,988, the entire contents of which are incorporated herein by reference); Dade Behring signal amplification methods (e.g., U.S. Patent Nos. US6,121,001, US6,110,677, US5,914,230, US5,882,867, and US5,792,614, the entire contents of which are incorporated herein by reference); ligation enzyme chain reactions (e.g., Baranay Proc. Natl. Acad. Sci. USA 88,189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Patent No. US5,288,609, the entire contents of which are incorporated herein by reference).

[0110] In some implementations, nucleic acid detection reagents include one or more of primers and probes.

[0111] The term "primer" refers to a short nucleic acid (oligonucleotide) that acts as the starting point for the synthesis of a polynucleotide chain by a nucleic acid polymerase under suitable conditions. Polynucleotide synthesis and amplification reactions generally involve a suitable buffer, NTPs (dNTPs, rNTPs, or combinations thereof), and one or more optional cofactors, and are carried out at a suitable temperature. Primers generally include at least one target hybridization region that is at least substantially complementary to the target sequence. This region is typically about 15 to about 40 nucleotides in length. A "primer pair" refers to a forward and reverse primer (sometimes called 5' and 3' primers) that are complementary to the opposite strand of the target sequence and designed to amplify the target sequence. The forward and reverse primers are aligned at an amplifiable distance from each other on the target sequence, for example, about 10–5000 nucleotides or about 25–500 nucleotides.

[0112] The term "dNTPs" stands for deoxyribonucleoside triphosphate, and "rNTPs" stands for ribonucleoside triphosphate.

[0113] As used herein, “probe” means any molecule capable of selectively binding to a specific, intended target biomolecule (e.g., a nucleic acid sequence of interest that will be bound, captured, or hybridized by the probe).

[0114] In a fourth aspect of the present invention, a method for processing viscous biological samples is provided, comprising the following steps:

[0115] Aqueous solution I was used to liquefy viscous biological samples to obtain liquefied material;

[0116] The composition of the aqueous solution I is as defined in the first aspect of the present invention.

[0117] In this invention, "aqueous solution I" is a mixture of the components of component a, or it can also be referred to as component a mixture.

[0118] In some implementations, the processing method satisfies one or more of the following features:

[0119] The volume ratio of the aqueous solution I to the viscous biological sample is 1 to 5 times;

[0120] The liquefaction is carried out at 18℃~35℃ for 1~15min;

[0121] During the liquefaction process, the mixture is stirred once or multiple times.

[0122] In some implementations, the processing method satisfies one or more of the following features:

[0123] The volume ratio of the aqueous solution I to the viscous biological sample is 2 to 4 times;

[0124] The liquefaction is carried out at 18℃~30℃ for 1~t1 min, where t1 is an integer selected from 5~10;

[0125] During the liquefaction process, the mixture is mixed 2 to 3 times every 5 to 10 minutes on average.

[0126] In some embodiments, the volume ratio of the aqueous solution I relative to the viscous biological sample can be 1 to 5 times, or it can be selected from any one of the following volume ratios or any range of two volume ratios: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc., or it can be selected from any of the following volume ratio ranges: 1.5 to 4.5 times, 2 to 4 times, etc.

[0127] In some embodiments, the liquefaction is performed at 18°C ​​to 35°C for 1 to 15 minutes. Component a in this invention can achieve simple, rapid, and efficient liquefaction of viscous biological samples at room temperature (e.g., 18 to 35°C, further e.g., 20 to 30°C), without the need for special high-temperature heating treatment, and the liquefaction time is short (e.g., ≤15 minutes, further e.g., ≤10 minutes).

[0128] In some embodiments of the present invention, the liquefaction temperature is room temperature, further can be 18–35°C, even further can be 18–30°C, and even further can be 20–30°C. The liquefaction temperature can also be selected from any one of the following temperatures or a range consisting of any two of the following temperatures: 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc.

[0129] In some embodiments of the present invention, the liquefaction time is 1 to 15 minutes, and may also be selected from any of the following durations or any two durations: 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, etc. The liquefaction time can also be selected from any of the following time ranges: 1–10 min, 1–5 min, 2–15 min, 2–10 min, 3–15 min, 3–10 min, 4–15 min, 4–10 min, 5–15 min, 5–10 min, 3–10 min, 3–5 min, 1–t1 min (where t1 is an integer selected from 5–15 or 5–10, and t1 can also be selected from any of the aforementioned suitable time ranges (in minutes), for example, t1 can be an integer selected from 2–15, 2–10, 3–15, 3–10, 4–15, 5–10 or 3–5).

[0130] In some embodiments, the liquefaction system can be forcefully mixed during the liquefaction process, and the mixing can be performed once or multiple times, for example, once, twice, three times, four times, five times or more. In some embodiments, the mixing is performed 2 to 3 times on average every 5 to 10 minutes during the liquefaction process. The mixing method is not particularly limited and can be stirring, shaking (such as a vortex mixer), swaying (such as a shaker, manual shaking), or a combination thereof.

[0131] In some embodiments, the processing method further includes mixing the liquefied material with component b to prepare a sample preservation solution; wherein component b is citric acid or an aqueous solution of citric acid, and the concentration of citric acid in the sample preservation solution is 1% to 10% (w / v). Using component a and component b in combination, after sample liquefaction, adding component b can react to generate sodium citrate and adjust the pH of the sample system to a suitable range, which can protect nucleic acids in multiple ways, thereby increasing the preservation time of the sample obtained by liquefying component a. When using component b, citric acid can be added directly to the liquefied mixture. To better control the amount of citric acid added for more precise pH adjustment, it can also be added as an aqueous solution of citric acid.

[0132] In this invention, "sample preservation" is involved, and unless otherwise stated, its purpose includes at least minimizing the loss of nucleic acids.

[0133] After mixing component b with the liquefied material, a volumetric dilution was performed relative to either the liquefied material or component b. By properly controlling the initial concentration of component b and the volumetric dilution factor of either the liquefied material or component b, the concentration of citric acid in the sample preservation solution can be controlled within a suitable range, thereby controlling the pH of the sample preservation solution within a suitable range.

[0134] In some embodiments, the pH of the sample preservation solution is 7.2 to 8.0, for example, it can be any one of pH values ​​such as 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, or a range consisting of any two of the aforementioned pH values.

[0135] In some embodiments, the concentration of citric acid in the sample preservation solution is 1% to 10% (w / v), further 1.5% to 8% (w / v), even further 1.5% to 6% (w / v), even further 2% to 6% (w / v), and may also be selected from any of the following concentrations or a range consisting of any two of the following concentrations: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, etc., in units of mass-volume concentration % (w / v).

[0136] In some embodiments, component b is an aqueous solution of citric acid with a mass-volume concentration of 10% to 30% (w / v) (which may also be selected from any suitable concentration described in the first aspect above), and the volume ratio of component b relative to the viscous biological sample is 0.8 to 1.2 times (e.g., 0.8 times, 0.9 times, 1 time, 1.1 times, 1.2 times, etc.).

[0137] In some implementations, the processing method satisfies one or more of the following features:

[0138] The pH of the sample preservation solution is 7.2–8.0;

[0139] The concentration of citric acid in the sample preservation solution was 1.5% to 8% (w / v);

[0140] The b component is a citric acid aqueous solution with a mass-volume concentration of 10% to 30% (w / v), and the volume ratio of the b component relative to the viscous biological sample is 0.8 to 1.2 times.

[0141] The maximum preservation time of the sample preservation solution is 3 to 8 days.

[0142] In some implementations, the sample preservation solution can be stored for a maximum period of 3 to 8 days, and the type or content of nucleic acids can still be tested by nucleic acid amplification to obtain nucleic acid information in the sample. See Examples 2 and 3 below.

[0143] In some embodiments, the processing method further includes the step of amplifying or detecting the target nucleic acid in the liquefied product or sample preservation solution obtained in the fourth aspect of the present invention using a nucleic acid amplification reagent. That is, it is equivalent to performing extraction-free amplification or detection without adding additional nucleic acid extraction reagents.

[0144] In some implementations, the processing method satisfies one or more of the following features:

[0145] The amplification is either extraction-free amplification or extraction-free detection;

[0146] The target nucleic acid includes DNA, RNA, or a combination of DNA and RNA.

[0147] In some implementations, the target nucleic acid, including RNA, is used in the preservation, extraction-free amplification, or extraction-free detection of the aforementioned viscous biological samples. See Examples 2, 3, and 4 below.

[0148] In some implementations, the target nucleic acid in the aforementioned preservation, extraction-free amplification, or extraction-free detection of viscous biological samples includes at least DNA. See Examples 2 and 3 below. DNA is more stable than RNA; extensive experimental exploration by the inventors of this application has also revealed that the liquefaction combination product for viscous biological samples provided by this invention, after liquefaction treatment of viscous biological samples (with or without the addition of component b), has good effects on extraction-free amplification and extraction-free detection of DNA in the samples.

[0149] In a fifth aspect of the invention, the application of the liquefaction combination product of the viscous biological sample described in the first aspect of the invention, or the nucleic acid amplification system of the viscous biological sample described in the second aspect of the invention, or the nucleic acid detection system of the viscous biological sample described in the third aspect of the invention.

[0150] In some implementations, the following applications are provided:

[0151] (A1) The application of the liquefaction combination product of the viscous biological sample according to the first aspect of the present invention in the liquefaction of viscous biological samples, wherein the liquefaction combination product comprises the a component and may or may not contain the b component; or,

[0152] (A2) The application of the liquefaction combination product of the viscous biological sample described in the first aspect of the present invention as a nucleic acid extraction reagent for independent use, may or may not contain the b component;

[0153] "Self-use nucleic acid extraction reagent" means that no other nucleic acid extraction reagents other than the sputum liquefaction combination product are allowed to be added. That is, the liquefaction combination product of viscous biological samples described in the first aspect of the present invention can be used for nucleic acid extraction-free applications. In this case, the direct amplification capability of the liquefaction combination product provided by the present invention can be used for instant detection. In some embodiments, the self-use nucleic acid extraction reagent is a self-use viral nucleic acid extraction reagent.

[0154] (A3) The application of the liquefaction combination product of the viscous biological sample according to the first aspect of the present invention in the preservation of viscous biological samples, wherein the liquefaction combination product comprises component a and component b; or,

[0155] (A4) The application of the liquefaction combination product of the viscous biological sample according to the first aspect of the present invention, or the nucleic acid amplification system of the viscous biological sample according to the second aspect of the present invention, in the extraction-free amplification of viscous biological samples, wherein the liquefaction combination product includes the a component and may or may not contain the b component; in some embodiments, the liquefaction combination product includes the a component and the b component; or,

[0156] (A5) The application of the liquefaction combination product of the viscous biological sample according to the first aspect of the present invention, or the nucleic acid amplification system of the viscous biological sample according to the second aspect of the present invention, or the nucleic acid detection system of the viscous biological sample according to the third aspect of the present invention in the extraction-free detection of viscous biological samples, wherein the liquefaction combination product includes the a component and may or may not contain the b component; in some embodiments, the liquefaction combination product includes the a component and the b component.

[0157] In this invention, "extraction-free amplification" and "extraction-free detection" refer to allowing the processed sample obtained in the fourth aspect to be directly used for nucleic acid amplification or nucleic acid detection without adding any nucleic acid extraction reagents other than the sputum liquefaction combination product described above. When implementing the processing method in the fourth aspect, the liquefaction combination product of the viscous biological sample described in the first aspect of this invention can simultaneously perform a certain nucleic acid extraction function while completing the liquefaction of the viscous biological sample. Therefore, the processed material can be directly used for subsequent nucleic acid amplification or nucleic acid detection. In the application of (A4) or (A5), only component a can be used to complete the liquefaction and then directly perform nucleic acid amplification or nucleic acid detection. Alternatively, components a and b can be used in combination. Adjusting the pH value of the system can further reduce the adverse effects on nucleic acid stability.

[0158] In some embodiments of this application, the application is for non-diagnostic and non-therapeutic purposes; in this case, it is not directly used for diagnosis or treatment.

[0159] In some embodiments of this application, the application is for diagnostic or therapeutic purposes.

[0160] The liquefaction combination product for viscous biological samples described in the first aspect of this invention can be flexibly used in conjunction with different nucleic acid detection platforms, including but not limited to use with real-time quantitative PCR instruments or portable nucleic acid detection instruments (such as the iPonatic mobile molecular detection system). The iPonatic mobile molecular detection system is a nucleic acid detection system publicly launched by Sansure Biotech Inc., while the real-time quantitative PCR instrument is a commonly used instrument well-known to those skilled in the art.

[0161] In some implementations, in the applications of aspects (A2), (A4) or (A5), the application refers to use in conjunction with a real-time quantitative PCR instrument or a portable nucleic acid detection instrument.

[0162] The following are some specific examples.

[0163] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, 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.

[0164] In the specific embodiments described below, the measurement parameters involving 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.

[0165] Unless otherwise specified, "room temperature" in the following text refers to 20-30°C, such as 25°C.

[0166] In the following examples of the liquefaction combination products for viscous biological samples of the present invention, unless otherwise specified, no other components are added, including no nuclease inhibitors (no RNase inhibitors or DNase inhibitors). The liquefaction combination products for viscous biological samples provided by the present invention can achieve good extraction-free amplification or extraction-free detection results without the addition of nuclease inhibitors; however, it should be understood that the liquefaction products for viscous biological samples of the present invention also allow the addition of other auxiliary components, including but not limited to nuclease inhibitors (including but not limited to those mentioned above), to achieve further optimization purposes (such as further improved nucleic acid detection results); it is understood that the addition of nuclease inhibitors does not affect the functional performance of the liquefaction combination products for viscous biological samples of the present invention. Technical solutions obtained by the foregoing modifications and similar modifications are also within the protection scope of the present invention.

[0167] The following embodiments use sputum samples as an example to describe the effects of the liquefaction combination product, liquefaction treatment and liquefaction treatment, nucleic acid amplification system, nucleic acid detection system, extraction-free amplification and other schemes of the present invention for viscous biological samples. However, the viscous biological samples of the present invention are not limited to this, and other types of samples described above are also allowed to be used to achieve the same or similar implementation and comparative effects when sputum is used as a viscous biological sample.

[0168] In the following examples, unless otherwise specified, water is used as the solvent for the preparation of liquid reagents, specifically sterile purified water. For instance, sodium hydroxide solution refers to an aqueous solution of sodium hydroxide dissolved in water, and citric acid solution refers to an aqueous solution of citric acid dissolved in water.

[0169] In the following examples, the test values ​​for Ct are automatically generated based on the software analysis results.

[0170] In this paper, the detection method for Ct value is highly accurate and has a small testing error. According to experimental observations, the Ct obtained from multiple tests on the same sample has very high stability, with a deviation value not exceeding ±0.2.

[0171] Unless otherwise specified, the deviation of the Ct test values ​​in the following examples shall not exceed ±0.1 for three sampling tests of the same sample.

[0172] The Ct value represents the number of cycles required for the fluorescence signal in each reaction tube to reach a set threshold. The Ct value of each template has a linear relationship with the logarithm of the initial copy number of that template, as shown in the following formula.

[0173] Ct=-1 / lg(1+E x )·lgX0+lgN / lg(1+E x X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of amplified product when the fluorescence amplification signal reaches the threshold intensity. n is the number of cycles of the amplification reaction, and when the amplification reaches the threshold, n = Ct.

[0174] The higher the initial copy number, the smaller the Ct value. A standard curve can be constructed using standards with known initial copy numbers, where the horizontal axis represents the logarithm of the initial copy number and the vertical axis represents the Ct value. Therefore, once the Ct value of a sample is obtained, the initial copy number of that sample can be calculated from the standard curve.

[0175] Therefore, the value of Ct can reflect the concentration of the sample being detected. The lower the Ct value, the higher the concentration of nucleic acid in the sample, and the higher the Ct value, the lower the concentration of nucleic acid in the sample. A difference of 1 Ct value indicates a twofold difference in the concentration of nucleic acid, a difference of 2 Ct values ​​indicates a fourfold difference, and so on.

[0176] In the following examples, unless otherwise specified, citric acid or its solution is added within 5-10 minutes after the liquefaction of component a mixture of the present invention is completed. In the following examples, "component a mixture" refers to a mixture obtained by mixing the components of component a. In the following examples, unless otherwise specified, "component a mixture" corresponds to the aqueous solution I mentioned above and can be directly used for the liquefaction of viscous biological samples.

[0177] Example 1. Evaluation of the liquefaction effect of the liquefaction combination product of the present invention

[0178] 1.1. Comparison with the traditional sodium hydroxide method

[0179] Seven clinical sputum samples were selected and compared with the traditional sodium hydroxide method to test the liquefaction effect of the liquefaction combination product of this invention. Clinical sputum samples were selected according to the following method: visually, samples were categorized as yellow, gray, rust-colored, bloody, purulent, thick, and lumpy. In this example, sputum samples with a consistency of grade 3 were selected. The consistency grade classification of the sputum samples is as follows:

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] In this example, the reagents of the present invention are prepared as follows: the final concentrations of component a mixture are as follows: acetylcysteine ​​5% (w / v) (306.75 mM), sodium chloride 200 mM, potassium chloride 6 mg / mL (approximately equivalent to 80.54 mM), and sodium hydroxide 0.45% (w / v) (112.5 mM); component b is an aqueous solution of citric acid with a citric acid mass / volume percentage of 20% (w / v). The pH of component a mixture is approximately 12.5.

[0185] In this example, the processing method of the present invention is as follows: add 3 times the volume of sputum sample of component A mixture and mix thoroughly. Let it stand at room temperature for 5 minutes (mix 2-3 times during the period) and observe the liquefaction effect. After complete liquefaction, add 1 times the volume of sputum sample of citric acid aqueous solution and mix thoroughly. Store or wait for use.

[0186] Traditional sodium hydroxide method: Add 4 times the volume of 4wt% sodium hydroxide solution (approximately 1 mol / L, pH approximately 14) to the sputum sample and mix thoroughly. Heat at 80℃ for 30 minutes, mixing 2-3 times during this period. Observe the liquefaction effect at time points of 5 min, 10 min, 15 min, and 30 min.

[0187] To compare the liquefaction effects of the two methods, the comparison indicators included the presence or absence of visible sputum (clear mucus is permissible) in the liquefied mixture, the presence or absence of excessive mucus, and whether pipetting operations could be performed (a 10 μL pipette was used in this example). A result of "no" for all three indicators indicated complete liquefaction and a good liquefaction effect. The results are shown in Table 1.

[0188] Table 1. Comparison of liquefaction effects

[0189]

[0190] *In Table 1, “The method of the present invention in this example” describes the liquefaction effect achieved before the addition of citric acid.

[0191] Based on the above experiments, the results show that in the sputum liquefaction test, the liquefaction component (component a) in the liquefaction combination product of the present invention is more effective than the traditional sodium hydroxide method in liquefying sputum of common grade 3 consistency. The solution provided by the present invention has significant advantages over traditional methods due to its shorter operation time and lower environmental requirements.

[0192] 1.2. Synergistic Effect Verification

[0193] To better verify the synergistic effect among the components in the liquefied combination product of this invention, the following comparative experiment was designed.

[0194] Two clinical sputum samples were selected (labeled I and II, respectively). Clinical sputum samples were selected according to the following method: based on visual inspection, samples were selected that exhibited yellow, gray, rust-colored, bloody, purulent, thick, and clumpy characteristics. Sputum samples with a consistency of grade 3 were selected.

[0195] The liquefying agent for experimental group 1 (i.e., the mixture of component a) is the same as the mixture of component a and component b in "Preparation of the reagents of the present invention in this example" in Part 1.1 of this example. Specifically, in the mixture of component a, the concentration of acetylcysteine ​​is 5% (w / v), and the concentration of sodium hydroxide is 0.45% (w / v), i.e., 112.5 mM.

[0196] Liquefying agent for Experimental Group 2: 5% (w / v) carboxymethylcysteine ​​was used to replace the 5% (w / v) acetylcysteine ​​in component a of the mixture in Experimental Group 1. The types and concentrations of the remaining components were the same as those in component a of Experimental Group 1. In this example, the concentration of carboxymethylcysteine ​​in the liquefying agent was 5% (w / v), and the concentration of sodium hydroxide was 0.45% (w / v), i.e., 112.5 mM.

[0197] Liquefying agent for Experimental Group 3: 5% (w / v) ambroxol was used to replace the 5% (w / v) acetylcysteine ​​in component a of the mixture in Experimental Group 1. The types and concentrations of the remaining components were the same as those in component a of Experimental Group 1. In this example, the concentration of ambroxol in the liquefying agent was 5% (w / v), and the concentration of sodium hydroxide was 0.45% (w / v).

[0198] Liquefying agent for Experimental Group 4: 5% (w / v) bromhexine was used to replace the 5% (w / v) acetylcysteine ​​in the mixture of component a in Experimental Group 1. The types and concentrations of the remaining components were the same as those in the mixture of component a in Experimental Group 1. In this example, the concentration of bromhexine in the liquefying agent was 5% (w / v), and the concentration of sodium hydroxide was 0.45% (w / v).

[0199] Liquefying agent in Experimental Group 5: 5% (w / v) guanidine hydrochloride was used to replace the 5% (w / v) acetylcysteine ​​in component a of the mixture in Experimental Group 1. The types and concentrations of the remaining components were the same as those in component a of Experimental Group 1. In this example, the concentration of guanidine hydrochloride in the liquefying agent was 5% (w / v), and the concentration of sodium hydroxide was 0.45% (w / v).

[0200] Liquefying agent in Experimental Group 6: 5% (w / v) ammonium chloride was used to replace the 5% (w / v) acetylcysteine ​​in component a of the mixture in Experimental Group 1. The types and concentrations of the remaining components were the same as those in component a of Experimental Group 1. In this example, the concentration of ammonium chloride in the liquefying agent was 5% (w / v), and the concentration of sodium hydroxide was 0.45% (w / v).

[0201] Treatment method for experimental groups 1-6: Add 3 times the volume of sputum liquefaction agent of each experimental group to the sputum sample and mix thoroughly. Let it stand at room temperature for 10 minutes, mixing 2-3 times during the period. Observe the liquefaction at time points such as 5 minutes and 10 minutes.

[0202] The sodium hydroxide solutions in experimental groups 7–12 below are all aqueous solutions, and the percentage concentrations are all in mass-volume units % (w / v).

[0203] In experimental groups 7–12, the liquefaction agent was used for 10 minutes, and the mixture was stirred 2–3 times during the process. The liquefaction status was observed at time points such as 5 minutes and 10 minutes.

[0204] Experimental Group 7 Liquefaction Agent: 4 wt% sodium hydroxide solution (approximately 1 mol / L) was used. Three times the volume of the 4 wt% sodium hydroxide solution was added to the sputum sample and mixed thoroughly. The sample was then heated at 80℃ for 10 minutes, mixing 2-3 times during the process.

[0205] Experimental group 8 liquefaction agent: Add 3 times the volume of 1000mmol / L sodium hydroxide solution (about 4wt%) to the sputum sample, mix thoroughly, and treat at room temperature for 10 min, mixing 2-3 times during the process.

[0206] Experimental Group 9 liquefaction agent: Add 3 times the volume of 112.5 mmol / L sodium hydroxide solution to the sputum sample, mix thoroughly, and treat at room temperature for 10 min, mixing 2-3 times during the process. Difference from Experimental Group 1: Acetylcysteine ​​was omitted.

[0207] Experimental Group 10: Liquefaction agent: Add 3 times the volume of 306.75 mmol / L acetylcysteine ​​(approximately 5% (w / v)) to the sputum sample, mix thoroughly, and treat at room temperature for 10 min, mixing 2-3 times during the treatment. Difference from Experimental Group 1: Sodium hydroxide was omitted from Experimental Group 1.

[0208] Example 11 Liquefaction agent: Add 3 times the volume of 420 mmol / L sodium hydroxide solution (approximately 306.75 + 112.5 mM) to the sputum sample, mix thoroughly, and treat at room temperature for 10 min, mixing 2-3 times during the process. Difference from Experimental Group 1: In Experimental Group 1, acetylcysteine ​​was replaced with sodium hydroxide of equal molar concentration.

[0209] Experimental Group 12: Liquefaction agent: Add 3 times the volume of 420 mmol / L acetylcysteine ​​(approximately 6.85% (w / v)) to the sputum sample, mix thoroughly, and treat at room temperature for 10 min, mixing 2-3 times during the treatment. Difference from Experimental Group 1: In Experimental Group 1, sodium hydroxide was replaced with an equal molar concentration of acetylcysteine.

[0210] To compare the liquefaction effects of each experimental group, the comparison indicators included whether there was visible sputum in the liquefied mixture (the presence of clear mucus was permissible), whether there was a large amount of mucus, and whether pipetting operations could be performed (a 10 μL pipette was used in this example). A result of "no" for all indicators indicated complete liquefaction and a good liquefaction effect. The results can be found in Table 2.

[0211] Table 2.

[0212]

[0213]

[0214] Experimental results show that, based on the mixture of component a of this invention, when acetylcysteine ​​is replaced with other commonly used liquefying components such as carboxymethylcysteine ​​(experimental group 2), ambroxol (experimental group 3), bromhexine (experimental group 4), guanidine hydrochloride (experimental group 5) or ammonium chloride (experimental group 6), or when cysteine ​​or sodium hydroxide (experimental groups 9-12) is omitted, the liquefaction effect is severely deteriorated, and most of them have no liquefaction effect within a liquefaction time of 5-10 minutes.

[0215] Based on extensive research by the inventors of this application, liquefaction was performed using the traditional sodium hydroxide method (see Experiment 7). No liquefaction occurred after 5 minutes, and liquefaction began with a small probability (less than 20%) after 10 minutes. In Experiment 7 of this example, under heating conditions at 80℃, neither sample liquefied after 5 minutes or 10 minutes.

[0216] Using the traditional method, the sodium hydroxide concentration (experimental group 8, approximately 1000 mmol) was used for liquefaction at room temperature, but no liquefaction effect was observed within a liquefaction time of 5-10 minutes.

[0217] 1.3. Investigation of the types and concentrations of each component in the liquefied component (component a)

[0218] Prepare the liquefying agent according to the formulas shown in Table 3.

[0219] Liquefying agent for experimental group 1: The composition is the same as the component a mixture in section 1.1 of Example 1. The operation steps, operation parameters and liquefaction effect test methods are the same as those for experimental group 1 in section 1.2 of Example 1.

[0220] The liquefying agents in experimental groups 2-19 replaced component a of the mixture in experimental group 1, and the operating procedures, operating parameters, and liquefaction effect testing methods were the same as those in experimental group 1 in this example.

[0221] Treatment methods for each experimental group: Add 3 times the volume of sputum liquefaction agent of each experimental group to the sputum sample and mix thoroughly. Let it stand at room temperature for 30 minutes, mixing 2-3 times during the period. Observe the liquefaction at different time points such as 5 min, 10 min, 15 min, 25 min, and 30 min. After the liquefaction is complete, add 1 volume of 20% (w / v) citric acid aqueous solution of sputum and mix thoroughly. Store or wait for use.

[0222] To compare the liquefaction effects of each experimental group, the comparative indicators included the presence or absence of visible sputum (clear mucus was permissible), the presence or absence of excessive mucus, and whether pipetting was possible (a 10 μL pipette was used in this example). A score of "no" for all indicators indicated complete liquefaction and good liquefaction results. The results can be found in Table 4.

[0223] Table 3.

[0224]

[0225]

[0226] Table 4.

[0227]

[0228]

[0229] It should be noted that Table 4 shows the liquefaction effect after 5 min and 10 min. Experimental groups 4, 6, 8, 13, and 14 achieved complete liquefaction within 15-25 min. Experimental groups 5 and 9 showed no significant liquefaction effect even after 30 min.

[0230] According to experimental results, regarding the A-component mixture of the present invention, when the concentration of acetylcysteine ​​is 3% to 15% (w / v) and the concentration of the strong base (calculated as a monobasic base) is in the range of 50 to 1000 mM, the concentration of acetylcysteine ​​and the type and concentration of the strong base can be arbitrarily adjusted. For common grade 3 sputum, a good liquefaction effect can be achieved within 5-10 minutes, that is, the time to achieve full liquefaction is ≤ t, where t can be selected from 5 to 10 minutes. In addition, the liquefaction effect is extremely excellent when the concentration of acetylcysteine ​​is 4% to 10% (w / v) and the concentration of the strong base (calculated as a monobasic base) is in the range of 100 to 600 mM, and a good liquefaction effect can be achieved within 5 minutes.

[0231] Furthermore, the inventors of this application have discovered that if the concentration of acetylcysteine ​​is too high (e.g., 15% (w / v)), the liquefaction effect decreases, mainly due to the formation of a gel-like substance, which prevents the reagent and sample from being further mixed and liquefied. Therefore, there is a synergistic effect between acetylcysteine ​​and strong bases (such as sodium hydroxide and potassium hydroxide). Even increasing the concentration of the other component cannot achieve the combined effect described in this invention if one of them is omitted.

[0232] Example 2. Test on the preservation effect of DNA pathogens after sputum liquefaction

[0233] 2.1. Verification of the preservation effect of DNA pathogens after sputum liquefaction

[0234] Three DNA pathogen culture samples (Saintville Medical Laboratory Center, PCR-positive bacteria) with a viscosity equivalent to grade 3 of sputum sample were selected to study the stability of the samples after liquefaction treatment with the reagent of this invention.

[0235] Experimental Group 1. In this example, the reagents of the present invention were prepared as follows: the concentrations of component a in the mixture were 5% (w / v) (306.75mM) of acetylcysteine, 200mM of sodium chloride, 6mg / mL (approximately 80.5mM) of potassium chloride, and 0.45% (w / v) (112.5mM) of sodium hydroxide; component b was a 20% (w / v) citric acid solution; the solvents in both components a and b were sterile purified water.

[0236] Add 3 times the sample volume of component A mixture to each sample and mix thoroughly. Let it stand at room temperature for 5 minutes to fully liquefy, mixing 2-3 times during this period. Then add 1 times the sample volume of citric acid solution and mix thoroughly. Take samples for testing at 0, 3, and 8 days of storage. The pH value of the system after adding citric acid solution should be in the range of 7.2-7.8, and also in the range of 7.2-8.0.

[0237] Experimental Group 2: Add 3 times the sample volume of component a mixture to each sample and mix thoroughly. Let it stand at room temperature for 5 minutes to fully liquefy, mixing 2-3 times during the process. Add 1 times the sample volume of commercial preservation solution to each of the 3 liquefied samples and mix thoroughly. Take samples for testing at 0 days, 3 days and 8 days of storage.

[0238] Specifications and composition of commercial preservation solution: X1001, the buffer component includes guanidine salt, metal chelating agent, surfactant and nucleic acid protectant.

[0239] DNA testing

[0240] The changes in pathogen DNA content after 0, 3, and 8 days of storage using two different preservation methods were tested using quantitative real-time PCR. The test results can be found in [reference needed]. Figure 1-6 See Table 5. Figure 1 , 3 Figures 5 and 6 show the amplification curves of DNA pathogenic bacteria culture samples 1, 2, and 3 after 0 days, 3 days, and 8 days of storage, respectively, using the liquefied combination product of the present invention (experimental group 1). Figure 2 , 4 Figures 6 and 7 show the amplification curves of DNA pathogenic bacteria culture samples 1, 2, and 3 after 0 days, 3 days, and 8 days of preservation using commercial preservation solution (experimental group 2).

[0241] In each amplification curve, the vertical axis Rn represents the fluorescence intensity of the PCR amplification product in the nth cycle, and the horizontal axis represents the cycle number. The corresponding Ct value can be obtained from each amplification curve. In this invention, the change in Ct value over time obtained from the amplification curve reflects the stability of the nucleic acid concentration; the smaller the change in Ct, the better the preservation effect on the sample.

[0242] Table 5.

[0243]

[0244] The test results of the above experiments show that the samples of the three selected DNA pathogen cultures, after liquefaction treatment and preservation using the reagents of this invention, exhibited good stability. The Ct changes after 3 days of preservation did not exceed 0.7 (all three samples in Table 5 were less than 0.6), and the Ct changes after 8 days did not exceed 1.5. Furthermore, compared with commercial preservation solutions, the liquefaction combination product of this invention demonstrates superior preservation stability. Commercial preservation solutions showed Ct changes greater than 0.7 after 3 days and greater than 1.6 after 8 days. Therefore, the liquefaction combination product of this invention can effectively ensure the stability of pathogen DNA samples after liquefaction. The liquefaction component (component a, mainly including acetylcysteine ​​and sodium hydroxide) and the preservation component (component b, mainly citric acid) exhibit a good synergistic effect.

[0245] 2.2. Investigation of different citric acid concentrations

[0246] Experimental Group 1: The composition of component a is the same as that of Experimental Group 1 in 2.1. of Example 2, but the concentration of the citric acid solution is changed to 10% (w / v). The operating procedures, operating parameters, and liquefaction effect testing methods are the same as those of Experimental Group 1 in 2.1. of Example 2. The working concentration of citric acid is approximately 2% (w / v).

[0247] Experimental Group 2. Based on Experimental Group 1 in this example, the concentration of the citric acid solution was changed to 30% (w / v). The working concentration of citric acid is approximately 6% (w / v).

[0248] The same method as in Example 2, section 2.1 was used for quantitative real-time PCR detection and analysis. The test results are shown in Table 6.

[0249] According to the test results, under the same usage volume, citric acid solution concentrations of 10%–30% (w / v) all provide good preservation of the liquefied product of component a. Table 6 shows that samples preserved using the reagent of this invention exhibit good stability; the Ct change values ​​after 3 days of preservation did not exceed 0.3, and the Ct change values ​​after 8 days of preservation did not exceed 0.9. It is understood that the concentration of the citric acid solution is not limited to this; for example, a more concentrated citric acid solution can be used as component b, and the dosage can be appropriately reduced to ensure the pH of the entire system remains within a suitable range.

[0250] Table 6.

[0251]

[0252] 2.3. Investigation into changing the type of acid in component b

[0253] Two DNA pathogen culture samples were selected, and the selection method was the same as in section 2.1 of Example 2.

[0254] Experimental Group 1: The same component a as in Example 2, 2.1 was used, with acetylcysteine ​​concentration of 5% (w / v), sodium chloride molar concentration of 200 mM, potassium chloride mass concentration of 6 mg / mL (approximately 80.5 mM), and sodium hydroxide concentration of 0.45% (w / v) (112.5 mM); component b was replaced with 20% (w / v) hydrochloric acid solution; the solvent in both components a and b was sterile purified water.

[0255] Add 3 times the sample volume of component A mixture to each sample and mix thoroughly. Let stand at room temperature for 5 minutes (mix 2-3 times during this period). Then add 1 times the sample volume of 20% (w / v) hydrochloric acid solution and mix thoroughly. Test the pH of the system.

[0256] Experimental Group 2: The amount of hydrochloric acid added was changed to keep the pH of the entire system in the range of 7.5 to 8.0. The volume was made up with sterile purified water. The rest was the same as Experimental Group 1.

[0257] Experimental Group 3. Replace the hydrochloric acid solution in Experimental Group 2 with 30% (w / v) acetic acid solution, control the amount added to keep the pH of the whole system basically within the range of 7.8 to 8.2, and make up the volume with sterile purified water. The rest is the same as Experimental Group 1.

[0258] The same method as in Example 2.2.1 was used to perform real-time quantitative PCR detection and analysis on each experimental group to examine the changes in pathogen DNA content after 0, 3, and 8 days of storage. The results are shown in Table 7.

[0259] The test results show that replacing citric acid with other types of acids worsened the preservation effect of the liquefied product of component a of this invention. Specifically, for samples from the same source, in experimental group 1, the Ct value of DNA changed by more than 2.5 after 8 days of preservation. In experimental groups 2 and 3, it was difficult to detect signal values ​​after 8 days, making statistical analysis of nucleic acid Ct values ​​impossible.

[0260] Table 7.

[0261]

[0262] In Table 7, "No Ct" indicates that no signal value was detected, and the result is negative.

[0263] Example 3. Preservation effect test of RNA virus culture after sputum liquefaction

[0264] 3.1. Verification of the preservation effect of RNA virus culture after sputum liquefaction

[0265] Two RNA virus culture samples were selected (RNA virus quality control material was added to grade 3 sputum, and the content was determined by PCR) to study the stability of the samples after liquefaction treatment with the reagents of this invention.

[0266] Experimental Group 1. In this example, the reagents of the present invention were prepared as follows: the concentrations of component a in the mixture were 5% (w / v) acetylcysteine, 200 mM sodium chloride, 6 mg / mL (approximately 80.5 mM) potassium chloride, and 0.45% (w / v) (112.5 mM) sodium hydroxide; component b was a 20% (w / v) citric acid solution; the solvents in both components a and b were sterile purified water.

[0267] Add 3 times the sample volume of component A mixture to each sample and mix thoroughly. Let it stand at room temperature for 5 minutes to fully liquefy, mixing 2-3 times during this period. Then add 1 times the sample volume of citric acid solution and mix thoroughly. Take samples for testing at 0 days, 3 days and 8 days of storage.

[0268] Experimental Group 2: Add 3 times the sample volume of component a mixture to each sample and mix thoroughly. Let it stand at room temperature for 5 minutes to fully liquefy, mixing 2-3 times during the process. Add 1 times the sample volume of commercial preservation solution to each of the two liquefied samples and mix thoroughly. Take samples for testing at 0 days, 3 days and 8 days of storage.

[0269] The specifications and composition of the commercial preservation solution are the same as those in 2.1 of Example 2.

[0270] RNA detection

[0271] The changes in viral RNA content after 0, 3, and 8 days of storage using two different preservation methods were tested using quantitative real-time PCR. The test results can be found in [reference needed]. Figure 7-12 See Table 8. Figure 7 , 9 11 and 12 are the amplification curves of RNA virus culture samples 1, 2, and 3 after 0 days, 3 days, and 8 days of storage, respectively, using the liquefied combination product of the present invention (experimental group 1); Figure 8 , 10 Figures 1 and 12 show the amplification curves of RNA virus cultures 1, 2, and 3 after 0, 3, and 8 days of preservation using commercial preservation solution (experimental group 2), respectively. In each amplification curve, the vertical axis Rn represents the fluorescence intensity of the PCR amplification product in the nth cycle, and the horizontal axis represents the cycle number. The corresponding Ct value can be obtained from each amplification curve.

[0272] Table 8.

[0273]

[0274] The experimental results show that the two selected RNA virus culture samples, after liquefaction treatment, exhibited good stability after preservation using the reagents of this invention. The Ct values ​​after 3 days of preservation did not exceed 1 (all three samples in Table 8 were less than 0.9), and the Ct values ​​after 8 days of preservation did not exceed 2.4. Furthermore, the stability was essentially comparable to commercial preservation solutions. Therefore, the liquefaction combination product of this invention can effectively ensure the stability of viral RNA in liquefied samples.

[0275] 3.2. Investigation into changing the type of acid in component b

[0276] Two RNA virus culture samples were selected using the same method as in section 3.1. Experimental group 1: The same component a as in section 3.1 was used, with acetylcysteine ​​concentration of 5% (w / v), sodium chloride molar concentration of 200 mM, potassium chloride mass concentration of 6 mg / mL (approximately 80.5 mM), and sodium hydroxide concentration of 0.45% (w / v) (112.5 mM); component b was replaced with 20% (w / v) hydrochloric acid solution; the solvent in both components a and b was sterile purified water.

[0277] Add 3 times the sample volume of component A mixture to each sample and mix thoroughly. Let it stand at room temperature for 5 minutes to fully liquefy (mixing 2-3 times during this period). Then add 1 times the sample volume of 20% (w / v) hydrochloric acid solution and mix thoroughly.

[0278] Experimental Group 2: The amount of hydrochloric acid added was changed to keep the pH of the entire system in the range of 7.5 to 8.5. The volume was then made up with sterile purified water. The rest was the same as Experimental Group 1.

[0279] The same method as in Example 3.1 was used to perform real-time quantitative PCR detection and analysis on each experimental group to examine the changes in viral RNA content after 0, 3, and 8 days of storage. The results are shown in Table 9.

[0280] According to the test results, it is evident that replacing citric acid with other types of acids significantly worsened the preservation effect of the liquefied product of component a of this invention. In experimental group 1, for the same sample, after 3 and 8 days of preservation, the Ct values ​​of RNA in the sample were difficult to detect, making statistical analysis of nucleic acid Ct values ​​impossible.

[0281] Table 9.

[0282]

[0283] "No Ct" indicates that no signal value was detected, and the result is negative.

[0284] Example 4. Investigation of the effect on the extraction-free amplification system (introducing unpurified nucleic acid extracted by amplification).

[0285] 4.1. Validation of the effectiveness of the extraction-free amplification system (introducing unpurified nucleic acid extracted by amplification).

[0286] Two sputum samples were selected, and the selection method was the same as in section 1.1 of Example 1.

[0287] Experimental Group 1. In this example, the reagents of the present invention were prepared as follows: the concentrations of component a in the mixture were 5% (w / v) acetylcysteine, 200 mM sodium chloride, 6 mg / mL (approximately 80.5 mM) potassium chloride, and 0.45% (w / v) (112.5 mM) sodium hydroxide; component b was a 20% (w / v) citric acid solution; the solvents in both components a and b were sterile purified water.

[0288] Add 3 times the sample volume of component A mixture to each sample and mix thoroughly. Let it stand at room temperature for 5 minutes to fully liquefy, mixing 2-3 times during this period. Then add 1 times the sample volume of citric acid solution and mix thoroughly (pH value approximately 7.8) to obtain the liquefied product. Do not use nucleic acid extraction reagents. Use the liquefied product directly as a matrix to dilute the RNA virus culture sample 100 times before performing amplification and detection by real-time PCR.

[0289] Experimental group 2: RNA virus culture samples were diluted 100-fold with physiological saline as a matrix and then amplified and detected by real-time PCR.

[0290] Experimental Group 3. The step of adding citric acid solution was omitted from Experimental Group 1. Three times the sample volume of component a mixture was added to each sample and mixed thoroughly. The mixture was placed at room temperature for 5 minutes (mixed 2-3 times during this period) (pH value approximately 12) to obtain the liquefied product. The RNA virus culture sample was directly diluted 100 times without nucleic acid extraction reagent and then amplified and detected by real-time PCR.

[0291] Experimental Group 4. Direct amplification after liquefaction using the traditional sodium hydroxide method. Add 4 times the volume of 4wt% sodium hydroxide solution (pH approximately 14) to the sputum sample and mix thoroughly. Incubate at 80℃ for 30 minutes, mixing 2-3 times during this period to obtain the liquefied sample. Without using nucleic acid extraction reagents, directly dilute the RNA virus culture sample 100-fold and perform amplification and detection using quantitative real-time PCR.

[0292] Experimental Group 5. After liquefaction treatment using the traditional sodium hydroxide method, the same amount of citric acid solution was added. Three volumes of 4wt% sodium hydroxide solution (pH approximately 14) were added to the sputum sample and mixed thoroughly. The sample was then heated at 80℃ for 30 minutes, mixing 2-3 times during this period. One volume of citric acid solution (pH approximately 7.9) was then added and mixed thoroughly to obtain the liquefied sample. Without using nucleic acid extraction reagents, the RNA virus culture sample was directly diluted 100-fold and amplified and detected using quantitative real-time PCR.

[0293] The test results can be found in Table 10.

[0294] Table 10.

[0295] Experimental group 1 The liquefaction combination product of this invention 25.17 27.62 Experimental group 2 physiological saline 25.51 27.37 Experimental group 3 No added citric acid NO Ct NO Ct Experimental group 4 Direct amplification after liquefaction treatment using traditional sodium hydroxide method NO Ct NO Ct Experimental group 5 Traditional sodium hydroxide liquefaction followed by the addition of the same amount of citric acid for amplification NO Ct NO Ct

[0296] "No Ct" indicates that no signal value was detected, and the result is negative.

[0297] The test results show that after diluting the two selected samples, the samples treated with the reagents of this invention exhibited essentially the same detection accuracy as those diluted with physiological saline matrix when amplified using the extraction-free method of quantitative real-time PCR. This demonstrates that the liquefaction combination product provided in this invention has good compatibility with the downstream extraction-free quantitative real-time PCR amplification and detection method, exhibits no inhibitory effect, and is highly user-friendly for downstream applications.

[0298] It should be noted that samples treated with the liquefaction combination product of this invention can also be treated with nucleic acid extraction reagents for nucleic acid extraction and then subjected to PCR amplification. The results are significantly better than those obtained by using traditional liquefaction agents for nucleic acid extraction and PCR amplification.

[0299] The technical features of the above embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above embodiments and examples 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.

[0300] The above embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed and specific understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and alterations without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of the present invention. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A liquefaction combination product for viscous biological samples, characterized in that, It includes component a, and may or may not contain component b; Component a is an aqueous solution I or a mother liquor of the following components: acetylcysteine ​​4%~10% (w / v), sodium chloride 20 mM~500 mM, potassium chloride 50 mM~120 mM, and a strong base; wherein, as a monobasic base, the concentration of the strong base in the aqueous solution I is 60 mM~1000 mM; the strong base is an inorganic strong base, and the inorganic strong base is at least one of sodium hydroxide and potassium hydroxide; Component b is citric acid or an aqueous solution of citric acid.

2. The liquefaction combination product according to claim 1, characterized in that, Satisfies any one or any suitable combination of the following characteristics: The viscous biological sample is sputum, cervical mucus, nasopharyngeal swab, or oral swab; The concentration of acetylcysteine ​​in the aqueous solution I is 5%~10%; Based on a monobasic base, the concentration of the strong base in the aqueous solution I is 100 mM ~ 600 mM; The citric acid aqueous solution has a citric acid concentration of 10% to 30% (w / v).

3. The liquefaction combination product according to claim 2, characterized in that, It meets one or more of the following characteristics: The inorganic strong base is sodium hydroxide; Based on a monobasic base, the concentration of the inorganic strong base in the aqueous solution I is 112.5 mM ~ 550 mM; The concentration of sodium chloride in the aqueous solution I is 150 mM ~ 250 mM; The concentration of potassium chloride in the aqueous solution I is 60 mM ~ 100 mM; The citric acid aqueous solution has a citric acid concentration of 15% to 25% (w / v).

4. The liquefaction combination product according to claim 1 or 2, characterized in that, The aqueous solution I contains the following components: acetylcysteine ​​5%~10% (w / v), sodium chloride 150 mM~250 mM, potassium chloride 60 mM~100 mM, and an inorganic strong base 100 mM~550 mM, wherein the concentration of the inorganic strong base is calculated as a monobasic base.

5. A nucleic acid amplification system for viscous biological samples, characterized in that, The product includes a liquefaction combination of viscous biological samples as described in any one of claims 1 to 4, and also includes nucleic acid amplification reagents.

6. A nucleic acid detection system for viscous biological samples, characterized in that, The product includes a liquefaction combination of viscous biological samples as described in any one of claims 1 to 4, and also includes nucleic acid detection reagents.

7. A method for processing viscous biological samples, characterized in that, The steps include the following: Aqueous solution I was used to liquefy viscous biological samples to obtain liquefied material; The composition of the aqueous solution I is as defined in any one of claims 1 to 4.

8. The processing method according to claim 7, characterized in that, It satisfies one or more of the following characteristics: The volume ratio of the aqueous solution I to the viscous biological sample is 1 to 5 times; The liquefaction is carried out at 18℃~35℃ for 1~15 min; During the liquefaction process, the mixture is stirred once or multiple times.

9. The processing method according to claim 8, characterized in that, It satisfies one or more of the following characteristics: The volume ratio of the aqueous solution I to the viscous biological sample is 2 to 4 times; The liquefaction is carried out at 18℃~30℃ for 1~t1 min, where t1 is an integer selected from 5~10; During the liquefaction process, the mixture is stirred 2 to 3 times every 5 to 10 minutes on average.

10. The processing method according to claim 7, characterized in that, The processing method further includes mixing the liquefied material with component b to prepare a sample preservation solution; wherein, component b is citric acid or an aqueous solution of citric acid, and the concentration of citric acid in the sample preservation solution is 1%~10% (w / v).

11. The processing method according to claim 10, characterized in that, It meets one or more of the following characteristics: The pH of the sample preservation solution is 7.2~8.0; The concentration of citric acid in the sample preservation solution was 1.5% to 8% (w / v); The b component is a citric acid aqueous solution with a mass-volume concentration of 10%~30% (w / v), and the volume ratio of the b component relative to the viscous biological sample is 0.8~1.2 times. The maximum preservation time of the sample preservation solution is 3 to 8 days.

12. The processing method according to claim 7, characterized in that, It also includes the following step: using a nucleic acid amplification reagent to amplify or detect the target nucleic acid in the sample preparation obtained in any one of claims 7 to 11.

13. The processing method according to claim 12, characterized in that, It satisfies one or more of the following characteristics: The amplification is either extraction-free amplification or extraction-free detection; The target nucleic acid includes DNA, RNA, or a combination of DNA and RNA.

14. The use of the liquefaction composition product of any one of claims 1 to 4 in the liquefaction of viscous biological samples, wherein the liquefaction composition product comprises component a and may or may not contain component b; or, The application of the liquefaction combination product of any one of claims 1 to 4 in the preservation of viscous biological samples, wherein the liquefaction combination product comprises component a and component b; or, The application of the liquefaction combination product of the viscous biological sample according to any one of claims 1 to 4, or the nucleic acid amplification system of the viscous biological sample according to claim 5, in the extraction-free amplification of viscous biological samples, wherein the liquefaction combination product includes component a, and may or may not contain component b; or, The application of the liquefaction combination product of any one of claims 1 to 4, the nucleic acid amplification system of the viscous biological sample of claim 5, or the nucleic acid detection system of the viscous biological sample of claim 6 in the extraction-free detection of viscous biological samples, wherein the liquefaction combination product includes the a component and may or may not contain the b component.

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