CN114107442B

CN114107442B - Viscous biological sample liquefaction release combination product, kit, liquefaction release method and nucleic acid extraction, amplification and detection method

Info

Publication number: CN114107442B
Application number: CN202210098137.XA
Authority: CN
Inventors: 邓中平; 陈诗谣; 邓勇; 刘佳; 戴立忠
Original assignee: Sansure Biotech Inc
Current assignee: Sansure Biotech Inc
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-05-27
Anticipated expiration: 2042-01-27
Also published as: CN114107442A

Abstract

The invention relates to a viscous biological sample liquefaction release groupA composite product comprising a liquefaction component and a nucleic acid release component: the liquefied component comprises guaifenesin and strong base; the nucleic acid release component comprises a component i) and/or a component ii), wherein the component i) comprises 0.1% -2% of Tween 20, 0.1% -3% of Triton X-100, 0.1% -3% of ethyl phenyl polyethylene glycol and 20 mM-1M of Na⁺And/or K⁺50mM to 1.25M strong base, an adsorbent and an aqueous solvent; component ii) comprises 0.01 to 0.5mM surfactant, 0.01 to 2% dodecylbenzene sulfonate, 50mM to 1.2M Na⁺And/or K⁺0.05% -1% of ethanol and third strong base. The nucleic acid releasing component and the liquefying component are matched for use, and the release effect on RNA and DNA is better.

Description

Viscous biological sample liquefaction release combination product, kit, liquefaction release method and nucleic acid extraction, amplification and detection method

Technical Field

The invention relates to the technical field of biological sample processing, in particular to a viscous biological sample liquefaction and release combination product.

Background

With the rapid development of fluorescent quantitative PCR and multiplex PCR technologies in the field of pathogen detection, the need for detecting whether a corresponding pathogen species is infected by a sputum sample type is increasing. However, sputum samples have the characteristics of high viscosity, high protein content and complex components, and contain mucin and other proteins (such as immune protein), enzymes, exfoliated cells, microorganisms and other inhalation impurities, so that direct detection is inconvenient, and clinical detection of the sputum samples requires liquefaction of the sputum first.

Common sputum liquefaction methods are sodium hydroxide method, DTT (dithiothreitol) method and protease method. The sodium hydroxide method is the most common sputum liquefaction method, the method is simple, the sputum is liquefied by using sodium hydroxide solution with a certain concentration as a main component at 60-80 ℃ (or under the room temperature condition), and when the method is used for nucleic acid detection, the strong alkaline environment of the method is easy to cause nucleic acid loss. The principle of the protease method is to digest mucus mucin by using protease, and the method has the advantages of long time consumption of enzymolysis reaction, high cost of the needed protease and low digestion efficiency of the mucin. The DTT (dithiothreitol) method is the most commonly used sputum liquefaction method at present, and utilizes DTT (dithiothreitol) containing sulfhydryl (-SH) to break down viscous main component mucin in sputum, uses PBS buffer solution (phosphate buffer solution) to provide physiological buffer, and usually adds ethanol and the like to fix cells. The method is long in time consumption, the used DTT (dithiothreitol) is high in cost and poor in DTT stability, the DTT needs to be stored under a low-temperature condition, and meanwhile, the DTT has certain toxicity and is not suitable for clinically processing sputum samples in a large scale. In addition, the sputum sample has high viscosity, so that the problem of uneven mixing is easily caused in the conventional liquefaction treatment process. Nucleic acids (especially RNA) in sputum samples are extremely unstable and are usually degraded under greenhouse conditions for hours. However, in clinical detection, nucleic acid in a sputum sample cannot be timely processed and detected, so that it is necessary to invent a method capable of quickly and sufficiently liquefying sputum and effectively protecting nucleic acid in the sample.

Many reports on methods for sputum liquefaction and nucleic acid protection have been published. However, these methods generally have the problems of complicated components, high cost, complicated operation, insufficient sample mixing and liquefaction, etc., and how to efficiently release nucleic acids from the liquefied sample is also a problem to be solved in the art.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is an object of the present invention to provide a viscous biological sample liquefaction release combination product comprising a liquefaction component and a nucleic acid release component:

the liquefaction component comprises guaifenesin and a strong base (labeled as the first strong base);

the nucleic acid releasing component is a composition represented by component i), a composition represented by component ii) or a composition comprising components i) and ii):

component i) comprises: 0.1-2% (v/v) Tween 20, 0.1-3% (v/v) Triton X-100, 0.1-3% (v/v) ethylphenyl polyethylene glycol, and 20-1 mol/L Na⁺And/or K⁺50 to 1.25 mol/L of a strong base (b)Labeled as a second strong base), an adsorbent (labeled as a first adsorbent, which may preferably be a Chelex resin), and an aqueous solvent;

component ii) comprises: 0.01 mmol/L-0.5 mmol/L surfactant, 0.01% -2% (w/v) dodecylbenzene sulfonate, 50 mmol/L-1.2 mol/L Na⁺and/K⁺0.05% -1% (v/v) ethanol and 100 mmol/L-1.25 mol/L strong base (labeled as third strong base), optionally also comprising (with or without) an adsorbent (labeled as second adsorbent, preferably Chelex resin).

The strong base (first strong base) in the liquefaction component, the second strong base, and the third strong base in the nucleic acid release component are each independently the same as or different from each other.

It is a further object of the present invention to provide a kit comprising a combination product as described above.

Still another object of the present invention is to provide a method for liquefying and releasing a viscous biological sample, comprising the steps of:

1) mixing a viscous biological sample with the liquefaction component as described above to obtain a first mixture;

2) incubating the first mixture to obtain a second mixture;

3) mixing the second mixture with a nucleic acid releasing component as described above and releasing the nucleic acid in the sample.

Still another object of the present invention is to provide a method for extracting nucleic acid from a viscous biological sample, comprising the steps of:

releasing the nucleic acids from the viscous biological sample using the method described above;

extracting the released nucleic acids using a nucleic acid extracting agent.

It is still another object of the present invention to provide a method for amplifying a nucleic acid in a viscous biological sample, comprising the following step 1), with or without the following step 2), and comprising the following step 3):

1) releasing the nucleic acids from the viscous biological sample using the method described above;

2) extracting the released nucleic acids using a nucleic acid extracting agent;

3) amplifying the nucleic acid using a nucleic acid amplification agent.

It is still another object of the present invention to provide a method for detecting nucleic acid in a viscous biological sample, comprising the following step 1), with or without the following step 2), with or without the following step 3), and comprising the following step 4):

2) extracting the released nucleic acids using a nucleic acid extracting agent;

3) amplifying the nucleic acid using a nucleic acid amplification agent;

4) detecting the nucleic acid using a nucleic acid detecting agent.

The combined product provided by the invention can rapidly liquefy the viscous biological sample, so that the viscosity of the sample is reduced, the subsequent operation is easy to carry out, the operation can be carried out at room temperature, the requirements on the operation conditions are not strict, and the subsequent nucleic acid detection is not adversely affected. The provided nucleic acid release component can be used in combination with a liquefaction component and has better release effects on RNA and DNA respectively.

The viscous biological sample liquefaction release combination product provided by the invention also has better compatibility, and has no adverse effect on subsequent nucleic acid detection, so that a hands-free amplification system can be compatible, and subsequent operations such as nucleic acid amplification, detection and the like can be directly carried out under the condition of no nucleic acid purification operation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing amplification curves obtained by rapid detection of human genomes by each group in one embodiment of the present invention; a is an experimental group; b is a protease method; c is a sodium hydroxide method; d is DTT method; e is an acetylcysteine method;

FIG. 2 is a graph showing the amplification curves obtained by the detection of the groups of respiratory syncytial viruses in one embodiment of the present invention; a is an experimental group; b is a protease method; c is a sodium hydroxide method; d is DTT method; e is an acetylcysteine method;

FIG. 3 is a graph showing the effect of the present invention on a hands-free amplification system (nucleic acid extracted by amplification is not purified); a is the result of the sample matrix No. 1 influencing the test amplification curve; b is the result of the sample matrix No. 2 influencing the test amplification curve;

FIG. 4 shows the results of a sample stability test after liquefaction of a reagent according to one embodiment of the present invention; a is an experimental group RNA stability curve; b is an experimental group DNA stability curve; c is a control group RNA stability curve; d is a control group DNA stability curve;

FIG. 5 shows the results of the detection of respiratory viruses after storage according to one embodiment of the present invention; a is sample No. 1, and the sample is preserved by adopting the example preservation component provided by the application; b is No. 1 sample, and is preserved by adopting commercial preservation solution; c is sample No. 2, preserved using the example preservation components provided herein; d is No. 2 sample, and is preserved by adopting commercial preservation solution;

FIG. 6 shows the results of the preservation of gonococci in one embodiment of the present invention; a is sample No. 1, and the sample is preserved by adopting the example preservation component provided by the application; b is No. 1 sample, and is preserved by adopting commercial preservation solution; c is sample No. 2, preserved using the example preservation components provided herein; d is No. 2 sample, and is preserved by adopting commercial preservation solution;

FIG. 7 shows the results of a cell sample after storage according to an embodiment of the present invention; a is sample No. 1, and the sample is preserved by adopting the example preservation component provided by the application; b is No. 1 sample, and is preserved by adopting commercial preservation solution; c is sample No. 2, preserved using the example preservation components provided herein; d is No. 2 sample, and is preserved by adopting commercial preservation solution;

FIG. 8 shows the results of different samples after being released by liquefaction in one embodiment of the present invention; a is an RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a No. 2 sample human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is a sample No. 2 gonococcus DNA amplification curve; the corresponding Ct position of the reagent of the application is marked in the figure;

FIG. 9 shows the results of testing different samples after being released from the liquefied storage according to one embodiment of the present invention; a is the RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a sample No. 2 human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is a sample No. 2 gonococcus DNA amplification curve;

FIG. 10 shows the results of different samples after being released by liquefaction in one embodiment of the present invention; a is the RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a No. 2 sample human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is a sample No. 2 gonococcus DNA amplification curve;

FIG. 11 shows the results of testing different samples after being released from liquefied storage according to one embodiment of the present invention; a is an RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a No. 2 sample human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is a sample No. 2 gonococcus DNA amplification curve;

FIG. 12 is a test amplification curve of the released reagent (formulation 10-2) of one embodiment of the present invention against sputum samples containing respiratory virus (A), gonococcus (B) and human cells (C);

FIG. 13 is a test amplification curve of the released reagent (formulation 10-3) of one embodiment of the present invention against sputum samples containing respiratory virus (A), gonococcus (B), and human cells (C);

FIG. 14 is a test amplification curve of the released reagent (formulation 10-4) of one embodiment of the present invention against sputum samples containing respiratory virus (A), gonococcus (B), and human cells (C);

FIG. 15 is a test amplification curve of the released reagent (formulation 12-2) of one embodiment of the present invention against sputum samples containing respiratory virus (A), gonococcus (B), and human cells (C);

FIG. 16 is a test amplification curve of the released reagent (formulation 12-3) of one embodiment of the present invention against sputum samples containing respiratory virus (A), gonococcus (B), and human cells (C);

FIG. 17 is a test amplification curve of the released reagent (formulation 12-4) of one embodiment of the present invention against sputum samples containing respiratory virus (A), gonococcus (B) and human cells (C).

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein, the term "and/or", "and/or" is selected to encompass any of two or more of the associated listed items, as well as any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all combinations of the associated listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or/and", "and/or", it should be understood that, in the present application, the technical solutions definitely include the technical solutions all connected by "logic and", and also the technical solutions all connected by "logic or". For example, "A and/or B" includes A, B and A + B. For example, the embodiments of "a, and/or, B, and/or, C, and/or, D" include any of A, B, C, D (i.e., all embodiments using a "logical or" connection), any and all combinations of A, B, C, D, i.e., any two or any three of A, B, C, D, and four combinations of A, B, C, D (i.e., all embodiments using a "logical and" connection).

In the present invention, the terms "plurality", and the like mean 2 or more in number unless otherwise specified. For example, "one or more" is 1 or 2 or more in number, and may be one, two, three or more.

As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

In the present invention, "preferably", "better" and "preferable" are only embodiments or examples with better description, and it should be understood that the scope of the present invention is not limited by them. If multiple 'preferences' appear in one technical scheme, if no special description exists, and no contradiction or mutual restriction exists, each 'preference' is independent.

In the present invention, "further", "still further", "specifically" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of the present invention.

In the present invention, "optionally" is not specifically defined, and may be absent or arbitrarily selected. If multiple optional items are present in one technical scheme, if no special description is provided, and no contradiction or mutual restriction exists, each optional item is independent.

In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one selected from the two juxtapositions "present" or "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent. For example, "optionally including" means including or not including.

The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The present invention relates to concentration values, which include fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may be allowed to fluctuate within 0.1%. For values that are larger or do not require more fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, may allow fluctuations within the range of. + -. 1%, + -2%, + -5%, etc. The molecular weight is referred to, allowing the meaning to include fluctuations of ± 10%.

In the present invention, the term "room temperature" generally means 4 ℃ to 35 ℃, preferably 20 ℃. + -. 5 ℃. In some embodiments of the invention, room temperature is 20 ℃ to 30 ℃.

In the present invention, the terms "first", "second", "third", etc. in the "first aqueous solvent", "second aqueous solvent", "first buffer component", "second buffer component", "third buffer component", "first strong base", "second strong base", "third strong base", "first adsorbent", "second adsorbent", "third adsorbent", "first mixture", "second mixture", etc. are used for descriptive purposes only for the purpose of distinction, and are not to be construed as indicating or implying a relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical feature indicated. Also, "first," "second," "third," etc. are for non-exhaustive enumeration description purposes only and should not be construed as constituting a closed limitation to the number. For example, in the present invention, the first strong base, the second strong base, and the third strong base are all strong bases, and may be the same or different, and the "first", "second", and "third" are merely used as distinguishing marks for distinguishing strong bases used in different embodiments. The molar concentration of the strong base in the present invention refers to the concentration of the hydroxide ion supplied, unless otherwise specified. When a specific strong base is defined, such as the molar concentration of sodium hydroxide, the specific definition controls. Such as 1M strong base, refers to a base having the ability to provide 1M hydroxide ion; 1M sodium hydroxide or other strong base species means that the strong base species has a molarity of 1M, and if a molecule of strong base carries more than one hydroxide ion, the ability to provide a molarity of hydroxide ions may exceed 1M.

The "water" referred to in the present invention may be, independently of each other, distilled water, purified water, filtered water, deionized water, or the like; each independently is preferably a nucleic acid-free water, and more each independently is preferably a nuclease-free water.

In the present invention, m/v represents a mass-to-volume ratio, and% (m/v) represents a mass percentage contained in a mixed system of a certain volume. For example, a concentration of 5% (m/v) of substance A in the mixed system means that 5g of substance A is contained per 100 ml of the mixed system.

In the present invention, mmol/L, mM represents millimoles per liter and may be used interchangeably. mol/L, M each represents moles per liter and may be used interchangeably.

In the present invention, the concentrations or amounts of the respective components in the liquefied component, if not specifically defined, refer to the final concentrations or final amounts in the liquefied component, which correspond to the final concentrations or final amounts in the liquefied component constituting the liquefying agent.

In the present invention, the concentrations or amounts of the respective components in the releasing component, if not specifically defined, refer to the final concentrations or final amounts in the releasing component, which correspond to the final concentrations or final amounts in the releasing agent constituted by the releasing component.

In the present invention, the concentrations or amounts of the respective components in the preserving component, if not specifically defined, refer to the final concentrations or final amounts in the preserving component, which correspond to the final concentrations or final amounts in the preserving agent constituted by the preserving component.

In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. The citation referred to herein is incorporated by reference in its entirety for all purposes unless otherwise in conflict with the present disclosure's objectives and/or technical solutions. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art features are also incorporated by reference into the present application, but the present invention is not limited to the embodiments. It should be understood that where the citation conflicts with the description herein, the application will control or be adapted in accordance with the description herein.

One aspect of the present invention relates to a viscous biological sample liquefaction release combination product comprising a liquefaction component and a nucleic acid release component:

the liquefaction component comprises guaifenesin and a strong base (noted as the first strong base);

the nucleic acid releasing component is a composition shown as component i), a composition shown as component ii) or a composition comprising component i) and component ii):

component i) comprises: 0.1-2% (v/v) Tween 20, 0.1-3% (v/v) Triton X-100, 0.1-3% (v/v) ethylphenyl polyethylene glycol, and 20-1 mol/L Na⁺And/or K⁺50 mmol/L-1.25 mol/L of strong base (marked as second strong base), adsorbent (marked as first adsorbent) and aqueous solvent (marked as second solvent);

component ii) comprises: 0.01mmol/L to 0.5mmol/L surfactant, 0.01% to 2% (w/v) dodecylbenzene sulfonate (independently preferably 0.01% to 1% (w/v)), 50mmol/L to 1.2mol/L Na⁺and/K⁺(independently preferably 60 mmol/L-1 mol/L), 0.05% -1% (v/v) ethanol, 100 mmol/L-1.25 mol/L strong base (marked as third strong base, independently preferably 150 mmol/L-1.25 mol/L), and optionallyComprising an adsorbent (denoted as second adsorbent).

The first strong base in the liquefaction component, the second strong base in the nucleic acid releasing component, and the third strong base are independent of each other, and may be the same as or different from each other.

In some embodiments, both guaifenesin and strong base are included in a single package.

In some embodiments, the liquefied component corresponds to a single package.

In some embodiments, the release component corresponds to a single package.

In some embodiments, the component i) corresponds to an individual package.

In some embodiments, said component ii) corresponds to one individual package.

In the present invention, "combination product" means that the components are not all mixed together, and one or more of the components may be separately packaged.

In the present invention, the terms "biological sample", "sample" and the like refer to an animal sample; tissue or organ, tissue lysate which may be from an animal (preferably including at least mammals, e.g. primates, including humans); cells (cells in a subject, taken directly from a subject, or maintained in culture or from a cultured cell line), cell lysate (or lysate fraction), or cell extract; a solution containing one or more molecules derived from cells, cellular material, or viral material (e.g., polypeptides or nucleic acids); or a solution containing naturally or non-naturally occurring nucleic acids that are or can be assayed as described herein. In some embodiments, the sample contains nucleic acids. The sample may also be any bodily fluid or excreta containing one or more cells, cellular components, or nucleic acids, including but not limited to cells, nuclei, or cell-free nucleic acids. In particular, the biological sample of the present invention is preferably derived from a body fluid, including a liquid, semi-solid, aerated liquid, liquid-gas mixture, etc. from an animal. Such bodily fluids may include, but are not limited to, saliva, sputum, serum, plasma, blood, urine, mucus, sweat, tears or other ocular fluids, ear fluids, face (e.g., from blisters or sores), gastric or stomach fluids, fecal fluids, pancreatic fluids or fluids, semen, nursing or assay products, spinal fluids, liquid bone marrow or lymph fluids.

"viscous biological sample" refers to a biological sample having viscosity, particularly a viscous body fluid. Viscosity may result from the presence of a large amount of components such as mucins and polysaccharides (particularly mucopolysaccharides) or proteoglycans in the biological sample. One preferred viscous biological sample is sputum and/or cervical mucus. Examples of some viscous biological samples are nasopharyngeal swabs, buccal swabs, lavage fluids, and the like. "mucin" refers to any viscous protein that increases the viscosity of the cytoplasmic matrix surrounding a secreting cell. "sputum" refers to viscous material (usually from the respiratory tract) contained in, or expelled from, the nasal or oral cavity of a mammal.

Liquefied component

In the present invention, "liquefying agent" and "liquefying agent" have the same meaning and may be used interchangeably, and are a mixture of liquefying components as a single system. The different components of the "liquefaction component" may be separate reagents or may be non-separate components.

The liquefaction component is a viscous substance for degrading a viscous biological sample species, so that the viscosity of the sample is reduced, and subsequent operations are easy to perform. The liquefaction component adopted in the invention can be liquefied at room temperature, has no harsh requirements on operating conditions, does not influence subsequent nucleic acid detection, can be compatible with a hands-free amplification system, and has no obvious adverse effect on the storage time of nucleic acid in a sample.

In the case of treating a viscous biological sample with a liquefying reagent, it is necessary to be in an aqueous environment, and therefore it is necessary to provide a solvent, and preferably an aqueous solvent. However, it should be understood that the solvent may be additionally provided during the processing operation, and thus, the solvent is optional for the liquefied component. In some embodiments, the liquefaction component preferably further comprises an aqueous solvent, which is further preferably water or a buffer solution.

After the guaifenesin and the first strong base in the liquefaction component are mixed, whether other components (such as one or more of an aqueous solvent, an adsorbent (marked as a third adsorbent), rigid microparticles and the like) are added or not is selected according to the situation, so that a liquefaction reagent can be obtained and is used for liquefying the viscous biological sample.

Guaifenesin in the liquefaction component has an excellent liquefaction effect relative to conventional liquefaction reagents. As shown in example 1 of the present invention.

In some embodiments, the concentration of guaifenesin is 1 to 1 mol/L. Herein, the concentration of guaifenesin refers to the concentration in the liquefying agent, unless particularly limited.

In some embodiments of the invention, the concentration of guaifenesin in the liquefied component is in the range of 20mmol/L to 500 mmol/L.

In some embodiments of the invention, the concentration range of guaifenesin includes, by way of example and not limitation: 20 to 400mmol/L, 50 to 200mmol/L, 50 to 150mmol/L, etc.

In some embodiments of the invention, the concentration of guaifenesin includes, but is not limited to: 20mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 80mmol/L, 100mmol/L, 120mmol/L, 140mmol/L, 150mmol/L, 160 mmol/L, 180 mmol/L, 200mmol/L, 220 mmol/L, 240 mmol/L, 250 mmol/L, 260 mmol/L, 280 mmol/L, 300mmol/L, 320 mmol/L, 340 mmol/L, 350 mmol/L, 360 mmol/L, 380 mmol/L, 400mmol/L, 420 mmol/L, 450 mmol/L, 500mmol/L, etc.

In the liquefied component, a first strong base is adopted to provide a strong base environment for providing a strong base environment with pH being more than or equal to 10. The kind of the first strong base is not particularly limited as long as it can provide a sufficiently liquefied pH condition without adversely affecting the subsequent PCR.

In the present invention, the term "strong base" refers to a substance in which the anions ionized in an aqueous solution are all hydroxide ions. It may be a strong organic or inorganic base including, but not limited to, at least one 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, silver diammine hydroxide, and the like. In some preferred embodiments, the strong base is sodium hydroxide and/or potassium hydroxide.

In some embodiments of the invention, the first strong base in the liquefaction component is a monobasic base at a concentration of from 5mmol/L to 500 mmol/L. Here, the concentration of the first strong base refers to the concentration in the liquefying reagent, unless otherwise specified. Examples of the concentration range of the first strong base include, but are not limited to, 5mmol/L to 400mmol/L, 5mmol/L to 200mmol/L, 5mmol/L to 100mmol/L, 5mmol/L to 80mmol/L, 5mmol/L to 60mmol/L, 5mmol/L to 50mmol/L, 5mmol/L to 40mmol/L, 10mmol/L to 200mmol/L, 10mmol/L to 100mmol/L, 10mmol/L to 50mmol/L, 10mmol/L to 40mmol/L, etc. Examples of concentrations include, but are not limited to, 5mmol/L, 6 mmol/L, 7 mmol/L, 8 mmol/L, 9 mmol/L, 10mmol/L, 12 mmol/L, 15 mmol/L, 20mmol/L, 25mmol/L, 30mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L, 100mmol/L, 120mmol/L, 140mmol/L, 150mmol/L, 200mmol/L, 250 mmol/L, 300mmol/L, 350 mmol/L, 400mmol/L, 450 mmol/L, 500mmol/L, and the like. The technical features of this embodiment may be combined in other embodiments in a suitable manner.

In some embodiments of the invention, the first strong base in the liquefaction component is at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, choline, and the like.

In some embodiments of the invention, the first strong base in the liquefaction component is sodium hydroxide and/or potassium hydroxide. Further, the concentration of the first strong base is 5 mmol/L-500 mmol/L. Examples of concentration ranges and examples of specific concentrations can be found in the examples section above for monobasic bases, which can be combined in a suitable manner to form a combination of features.

In some embodiments, the first strong base in the liquefaction component is sodium hydroxide.

In some embodiments, the guaifenesin and the first strong base in the liquefied component are provided in a composition (co-packaged in one container). In this case, the concentration of guaifenesin in the liquefying agent is preferably 1 to 1mol/L, and the concentration of the first strong base in the liquefying agent is preferably 5 to 500mmol/L (more preferably sodium hydroxide). The concentrations of guaifenesin and the first strong base may each be selected from the examples of concentrations or concentration ranges set forth herein, and the concentrations between the two may be combined in any suitable manner to achieve the desired liquefaction.

In some preferred embodiments, the liquefaction component produces a liquefaction reagent comprising 100mmol/L guaifenesin and 10mmol/L sodium hydroxide.

Optionally including a solvent in the liquefied component; when included, the solvent is preferably an aqueous solvent; the aqueous solvent herein is referred to as a first aqueous solvent.

In some preferred embodiments, the liquefaction component consists of guaifenesin and a strong base (noted as the first strong base), a solvent (further preferably an aqueous solvent).

The term "aqueous solvent" as used herein is a solvent or solution containing water, and may be a single solvent consisting of pure water or a mixed solvent in which water is miscible with other solvents. Water-miscible solvents, including but not limited to: alcohol solvents (e.g., methanol, ethanol, propanol, isopropanol, polyethylene glycol, etc.). The aqueous solvent also allows for the inclusion of a salt component.

In some embodiments of the invention, the first aqueous solvent is water. Such as distilled water, purified water, filtered water, deionized water, etc.; preferably, the water is free of nucleic acid, more preferably free of nuclease.

In some embodiments of the present invention, the first aqueous solvent is a buffer component, denoted as the first buffer component.

The term "buffer solution," also referred to herein as a "buffer component," as used herein, refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffering properties of such liquids. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have the unlimited ability to maintain the pH of a solution or composition. Instead, they are generally capable of maintaining a pH within a specified range, such as pH6 to pH8, and further such as pH ≧ 10. Generally, Buffers are capable of maintaining a pH at their pKa and within the next logarithm (see, e.g., Mohan, Buffers, A guide for the preparation and use of Buffers in biological systems, CALBIOCHEM, 1999). Where the liquefaction component includes a buffer component, the buffer component (i.e., the first buffer component) is intended to maintain the pH of the liquefaction system in a strongly alkaline environment, such as a pH ≧ 10.

The liquefied component optionally includes an adsorbent, i.e., the adsorbent is an optional component, not required.

In some embodiments, the liquefaction component further includes an adsorbent, denoted as a third adsorbent. In some preferred embodiments, the third adsorbent is a chelating resin, further a Chelex resin. In some embodiments, the concentration of the third adsorbent in the liquefied component is 1% to 15% (w/v), such as 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., wherein the third adsorbent is preferably a chelating resin, further a Chelex resin.

The liquefaction component may also optionally include rigid microparticles. That is, rigid microparticles may be an optional component.

In some embodiments, the liquefaction component further includes rigid microparticles that may serve to accelerate blending.

In some embodiments, the rigid microparticles correspond to a single package.

The rigid microparticles may be made of any hard material, and the term "rigid" refers to the microparticles that are not usually significantly damaged during mixing/assisted mixing of the viscous biological sample and do not release components that interfere with subsequent processes (e.g., at least one of preservation, release, enrichment, amplification, and detection of nucleic acids). The material includes but is not limited to metal (which may be simple substance or alloy) or metal oxide, ceramic, glass, hard plastic, natural or artificial mineral composition, etc. In some preferred embodiments, the material of the rigid microparticles includes at least one of zirconia, silicon nitride, ceramsite, hard stainless steel, hard tungsten carbide, sintered corundum, and agate.

The rigid microparticles may be used to assist in blending the viscous biological sample and the viscous biological sample liquefaction composition, reducing processing time, and increasing processing efficiency.

In the present invention, the term "microparticle" may be a sphere, a nearly sphere, an ellipsoid, a column, a rod, a polyhedron (e.g., a cube), or an irregular shape, and is preferably a microsphere. The average particle diameter of the microparticles is preferably in the order of millimeter, for example, 0.01mm to 500mm, and may be 0.05mm, 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 20mm, 30mm, 40mm, 50mm, 100mm, 200mm, 300mm, 400mm, 500mm, and the like, and more preferably 0.01mm to 10 mm.

In some embodiments of the present invention, the amount of the rigid microparticles is 0.1 g/mL to 2g/mL (which is the final amount in the liquefied component, if not specifically limited). Examples of amounts include, but are not limited to, 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6 g/mL, 0.7g/mL, 0.8g/mL, 0.9g/mL, 1.0g/mL, 1.1g/mL, 1.2g/mL, 1.3g/mL, 1.4g/mL, 1.5g/mL, 1.6g/mL, 1.7g/mL, 1.8g/mL, 1.9g/mL, 2.0g/mL, and the like.

In some embodiments, the liquefaction component includes guaifenesin, a strong base, and rigid microparticles. In some preferred embodiments, the guaifenesin, the strong base and the rigid microparticles in the liquefaction component are provided in a mixed system comprising three (co-packaged in a container), and preferably also comprise a solvent, which is further preferably an aqueous solvent, more preferably water or a buffer solution. Wherein the content of guaifenesin is preferably 1 mmol/L-1 mol/L, the content of strong base is preferably 5 mmol/L-500 mmol/L (more preferably sodium hydroxide), and the content of rigid microparticles is preferably 0.1 g/mL-2 g/mL (more preferably zirconia beads).

In some preferred embodiments, 100mM guaifenesin, 10mM strong base (independently preferably sodium hydroxide), and 1g/mL rigid microparticles (independently preferably zirconia beads) are included in the liquefaction component.

In some preferred embodiments, the liquefaction component consists of guaifenesin and, a strong base, rigid microparticles, and an aqueous solvent.

In some embodiments, the liquefaction reagent comprised of liquefaction components includes (final concentrations): 100mM guaifenesin, 10mM sodium hydroxide and 1g/mL rigid microparticles (preferably zirconia beads) in water.

The components in the liquefied components can be packaged separately or can be packaged together.

In some embodiments, the liquefied component is a composition, packaged in a container. In some embodiments, one container contains guaifenesin and a strong base in the liquefied component and another container contains rigid microparticles. In some embodiments, the guaifenesin, the strong base, and the rigid microparticles are each packaged separately. In the present invention, the composition may be a solid (preferably a dry powder) or a liquid, or a state in between such as a gel state. In some embodiments, the composition is a solution. The solvent of the solution may be water, such as distilled water, purified water, filtered water, deionized water, etc.; preferably, the water is free of nucleic acid, more preferably free of nuclease.

In some embodiments, the liquefied component composition is a mixture and the rigid microparticles have a concentration of 0.1 g/mL to 2.0g/mL, such as 0.1 g/mL, 0.2 g/mL, 0.3 g/mL, 0.4 g/mL, 0.5 g/mL, 0.6 g/mL, 0.7g/mL, 0.8g/mL, 0.9g/mL, 1.0g/mL, 1.1g/mL, 1.2g/mL, 1.3g/mL, 1.4g/mL, 1.5g/mL, 1.6g/mL, 1.7g/mL, 1.8g/mL, 1.9g/mL, 2.0g/mL, and the like.

Release component

In the present invention, "release agent" refers to a separate system, which is a mixture of release components. The different components of the "delivery component" may be separate agents or may be separate components.

The components of the release composition may be mixed to provide a separate release agent for releasing nucleic acids from a viscous biological sample or a processed viscous biological sample.

"Release component" as used herein refers to a component for releasing nucleic acids, i.e.a component for effecting the release of nucleic acids. The term "release of nucleic acid" means that nucleic acid is released from the sample and is in an extractable/enriched/purified/detectable state. The release of nucleic acids is usually accompanied by lysis of cells and separation of components of impurities such as proteins, lipids, polysaccharides, etc. from nucleic acid components in a physical state, unlike a physiological state, which can be separated by a simple centrifugation, etc., and nucleic acids can be directly detected by some detection reagents.

component i) comprises: 0.1-2% (v/v) Tween 20, 0.1-3% (v/v) Triton X-100, 0.1-3% (v/v) ethylphenyl polyethylene glycol, and 20-1 mol/L Na⁺And/or K⁺50 mmol/L-1.25 mol/L of strong base (marked as second strong base), adsorbent (marked as first adsorbent) and aqueous solvent (marked as second aqueous solvent);

component ii) comprises: 0.01 mmol/L-0.5 mmol/L surfactant, 0.01% -2% (w/v) dodecylbenzene sulfonate, 50 mmol/L-1.2 mol/L Na⁺and/K⁺0.05% -1% (v/v) ethanol, 100 mmol/L-1.25 mol/L strong base (marked as third strong base), and optionally an adsorbent (also marked as second adsorbent); further comprising a solvent (preferably water).

The component ii) is released by: the cells are lysed, releasing the nucleic acids.

The component i) is released in the following manner: cell lysis, nucleic acid release and nucleic acid structure stability maintenance (RNA is easy to degrade).

When the component i) is used in admixture with the component ii), the cells are lysed, the nucleic acids are released and the structural stability of the nucleic acids is maintained.

In the component i), the concentration of tween 20 may be 0.1% to 2% (v/v), further may be 0.5% to 1.5% (v/v), and further may be 0.8% to 1.2% (v/v), for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, and the like in terms of volume percentage.

In the component i), the concentration of the triton X-100 may be 0.1% to 3% (v/v), further 0.2% to 2% (v/v), further 0.5% to 1.5% (v/v), further 0.8% to 1.2% (v/v), for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, etc., in terms of volume percentage.

In component i), the ethylphenylpolyethylene glycol is in a liquid state, and the molecular weight of the polyethylene glycol moiety is not particularly limited as long as it is in a liquid state or can be completely dissolved in component i). The concentration of the ethylphenylpolyethyleneglycol in component i) may be 0.1% to 3% (v/v), further 0.2% to 2% (v/v), further 0.5% to 1.5% (v/v), further 0.8% to 1.2% (v/v), for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, etc., in volume percentage.

In component i), Na⁺、K⁺The concentration of (b) may be, independently, 20 to 1mol/L, further 60 to 500mmol/L, further 60 to 200mmol/L, further 60 to 100mmol/L, and examples thereof may be, independently, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250 mmol/L, and 500 mmol/L. In one embodiment, Na⁺In a concentration of 100mM, K⁺Has a concentration of 80 mM. Na (Na)⁺、K⁺Each independently may be provided by a corresponding salt and/or base, such as NaCl, KCl, NaOH, KOH, and the like. In one embodiment, component i) comprisesThere was 100mM NaCl, 80mM KCl and 250mM NaOH. In some embodiments, Na is provided by a salt form⁺、K⁺(i.e., sodium salt, potassium salt, such as sodium chloride, potassium chloride, respectively), Na provided in salt form⁺、K⁺The concentration of (b) may be, independently, 60 to 500mmol/L, further 60 to 200mmol/L, further 60 to 100mmol/L, and examples thereof may be, independently, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250 mmol/L, and 500 mmol/L.

In component ii), the surfactant may be one or more of sarsantine, Sodium Dodecyl Sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and the like. In some preferred embodiments, the surfactant is sargentin. The concentration of the surfactant (preferably sanskantin) in the component ii) may be 0.01mmol/L to 0.5mmol/L, further may be 0.1mmol/L to 0.5mmol/L, and further may be 0.2mmol/L to 0.5 mmol/L. Specific examples thereof include 0.01mM, 0.05mM, 0.1mM, 0.15 mM, 0.2mM, 0.25mM, 0.3mM, 0.35 mM, 0.4mM, 0.45 mM, and 0.5 mM.

In component ii), the dodecylbenzene sulfonate may be a sodium salt. The concentration of the dodecylbenzene sulfonate in the component ii) may be 0.01% to 2% (w/v), further 0.1% to 1.5% (w/v), or 0.5% to 2% (w/v), further 0.1% to 1.5% (w/v), further 0.5% to 1.5% (w/v), further 0.8% to 1.2% (w/v). Specific examples include intervals of any one percentage or any two percentages of the following groups: 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc., in terms of mass-to-volume ratio. In some preferred examples of the invention, the concentration of dodecylbenzene sulfonate in component ii) may be 0.01% to 1% (w/v).

In component ii), Na⁺and/K⁺Preferably provided in the form of a salt (sodium and/or potassium salt), such as for example sodium chlorideAnd potassium chloride. Na (Na)⁺And K⁺The concentration in the component ii) may be 50 mM-1.2M, further 50 mM-1M, further 50 mM-500 mM, and further 80 mM-500 mM. Specific examples thereof include 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160 mM, 180 mM, 200mM, 300mM, 350 mM, 400mM, 450 mM, 500mM, 550 mM, 600mM, 650 mM, 700mM, 750 mM, 800mM, 850 mM, 900mM, 950 mM, 1M and the like. In some embodiments, the concentration of the sodium salt and/or potassium salt (e.g., sodium chloride, potassium chloride) in component ii) is 50 mM-200 mM, further 50 mM-150 mM, further 50 mM-120 mM, further 80 mM-120 mM. Specific examples thereof include 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160 mM, 180 mM, and 200 mM.

In the component ii), the concentration of ethanol may be 0.05% to 1% (v/v), further 0.1% to 0.8% (v/v), further 0.2% to 0.8% (v/v). Specific examples thereof include 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, 1% and the like in terms of volume percentage.

In the present invention, the first strong base, the second strong base, and the third strong base are all strong bases. A first strong base is included in the liquefaction component for providing an alkaline environment for liquefaction of the viscous biological sample. And a second strong base, a third strong base, is included in the nucleic acid releasing component to ensure release of the nucleic acid in a strongly alkaline environment. In the present invention, the first strong base, the second strong base, and the third strong base are each independently defined, may be the same or different, and are each independently selected from the "strong bases" defined in the present invention.

In some embodiments, the second strong base in component i) is sodium hydroxide and/or potassium hydroxide.

In some embodiments of the invention, the second strong base is a monobasic base (e.g., sodium hydroxide and/or potassium hydroxide) at a concentration of 50mM to 1.25M. Examples of concentration ranges include, but are not limited to, 50 mM-1M, 50 mM-800 mM, 50 mM-600 mM, 50 mM-500 mM, 50 mM-400 mM, 50 mM-200 mM, 100 mM-1M, 100 mM-800 mM, 100 mM-600 mM, 100 mM-500 mM, 100 mM-400 mM, 200 mM-800 mM, 200 mM-500 mM, 200 mM-400 mM, and the like. Examples of concentrations include, but are not limited to, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 140mM, 150mM, 200mM, 250mM, 300mM, 350 mM, 400mM, 450 mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1M, and the like. The technical features of this embodiment may be combined in other embodiments in a suitable manner.

In some embodiments of the invention, the second strong base is at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, choline, and the like.

In some embodiments of the invention, the second strong base is sodium hydroxide and/or potassium hydroxide. Further, the concentration of the second strong base in the component i) is 50 mM-1.25M, and further can be 50 mM-1M.

In some embodiments of the invention, the third strong base is a monobasic base (e.g., sodium hydroxide and/or potassium hydroxide) at a concentration of 100mM to 1.25M. Examples of concentration ranges include, but are not limited to, 100 mM-1M, 100 mM-900 mM, 100 mM-800 mM, 100 mM-600 mM, 100 mM-500 mM, 100 mM-400 mM, 150 mM-1M, 150 mM-800 mM, 150 mM-1600 mM, 150 mM-500 mM, 150 mM-400 mM, 200 mM-800 mM, 200 mM-500 mM, 200 mM-400 mM, and the like. Examples of concentrations include, but are not limited to, 100mM, 120mM, 140mM, 150mM, 200mM, 250mM, 300mM, 350 mM, 400mM, 450 mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1M, and the like. The technical features of this embodiment may be combined in other embodiments in a suitable manner.

In some embodiments of the invention, the third strong base is sodium hydroxide and/or potassium hydroxide. Further, the concentration of the third strong alkali in the component ii) is 150mM to 1.25M, and further can be 150mM to 1M.

In the present invention, the first aqueous solvent is one of the components of the liquefaction component and the second aqueous solvent is one of the components of component i). The first aqueous solvent and the second aqueous solvent are each independently defined, and each is independently selected from the "aqueous solvents" defined in the present invention, and may be the same or different.

In some embodiments of the invention, the second aqueous solvent is water. Such as distilled water, purified water, filtered water, deionized water, etc.; preferably, the water is free of nucleic acid, more preferably free of nuclease.

In some embodiments of the present invention, the second aqueous solvent is a buffer component, denoted as the second buffer component.

The first buffer component and the second buffer component are each independently defined and each independently selected from the "buffer components" defined in the present invention, and may be the same or different.

When the component i) contains a buffer component, the dosage ratio of the buffer component to the second strong base should be reasonably controlled to ensure that the nucleic acid can be effectively released in an alkaline environment. In some embodiments, in component i), the buffer component is Tris-HCl, and the second strong base is sodium hydroxide, wherein the molar amount of NaOH may be 1 to 5 times, and further may be 1.5 to 5 times, such as 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, and 5 times of Tris-HCl.

In some embodiments, component i) comprises a buffer component, preferably selected from the group consisting of: Tris-HCl, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, disodium hydrogen phosphate-citric acid buffer, more preferably 0.5mmol/L to 500mmol/L Tris-HCl, and the concentration of the Tris-HCl system may be 1mmol/L, 10mmol/L, 50mmol/L, 100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L or the like.

In some embodiments, the second aqueous solvent is Tris-HCl, preferably 0.5-500 mmol/L Tris-HCl.

The component i) should in principle also contain an adsorbent (also denoted as first adsorbent) in order to better achieve a liquefied release of the RNA virus sample.

The adsorbent can adsorb impurities through physical or chemical action, and interference of the impurities on subsequent detection is reduced. The adsorbent may be arbitrarily selected from suitable types without adversely affecting the liquefaction. The chemistry used to adsorb impurities includes, but is not limited to, chelation. The adsorbent can be resin or chelate, and can also be chelate resin. In some embodiments, the adsorbent is a resin. In some preferred embodiments, the adsorbent is a resin such as a polypropylene resin, a polyacrylic acid resin, a polyvinyl alcohol resin, or a chitosan resin, and more preferably, a chelate resin. In some preferred embodiments, the adsorbent is a Chelex resin.

The "adsorbent" in the present invention may be distinguished and labeled as "first adsorbent", "second adsorbent", "third adsorbent", corresponding to component i), component ii) and liquefied component, respectively, depending on the system to which it belongs. The three are each independently defined, each independently selected from the "adsorbents" defined in the present invention, and may be the same as or different from each other, and each independently is preferably a chelate resin, and further each independently is preferably a Chelex resin. The adsorbent is optional in both the liquefaction component, component ii), and is necessary for the release of RNA in component i). In some embodiments, the viscous biological sample liquefaction release combination product includes only the second sorbent and no first sorbent and no third sorbent. In some embodiments, the viscous biological sample liquefaction release combination product includes only the second sorbent and the third sorbent. In some embodiments, the viscous biological sample liquefaction release combination product includes only the first sorbent and the third sorbent.

In some embodiments, the concentration of the first adsorbent is 1% to 15% (w/v), e.g., 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., referring to the final concentration in the release component. In some preferred embodiments, the first adsorbent is a chelating resin, further a Chelex resin.

In some embodiments, the concentration of the second adsorbent is 0.1% to 15% (w/v), e.g., 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., referring to the final concentration in the release component. In some preferred embodiments, the second adsorbent is a chelating resin, further a Chelex resin.

In some embodiments, the concentration of the third adsorbent is 0.1% to 15% (w/v), such as 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., referring to the final concentration in the liquefied component. In some preferred embodiments, the third adsorbent is a chelating resin, further a Chelex resin.

The addition of an adsorbent (preferably a chelating resin) to the released components has an effect of improving the sensitivity of DNA detection.

The addition of an adsorbent, preferably a chelating resin, to the releasing component also has a key effect on the release of RNA. When RNA detection is carried out, reverse transcription is usually carried out, but the reverse transcription process is easily interfered by impurities such as protein, polysaccharide and the like, so that the reverse transcription efficiency of RNA is low, and subsequent amplification and detection cannot be carried out. By adding the adsorbent, impurities can be effectively adsorbed, and impurity interference is reduced or avoided, so that subsequent amplification and detection can be smoothly implemented.

In the component i), trehalose is optionally further contained. In some embodiments, the trehalose is at a concentration of 0.5mol/L to 1mol/L in component i), e.g., 0.5mol/L, 0.6mol/L, 0.7 mol/L, 0.8mol/L, 0.9 mol/L, 1mol/L, and the like. In some embodiments, the first adsorbent comprises 0.5mmol/L to 1mmol/L trehalose.

In some embodiments, component i) comprises: 0.5-1.5% (v/v) Tween 20, 1-2% (v/v) Triton X-100, 1-2% (v/v) ethylphenyl polyethylene glycol, and 100-300 mol/L Na⁺And/or K⁺、150mmol/L to 350 mol/L of a second strong base (such as sodium hydroxide and/or potassium hydroxide, for example), 1% to 10% (w/v) of an adsorbent (such as Chelex resin, for example), optionally trehalose, and an aqueous solvent (such as one or more of water, a buffer component, and the like, preferably 50mM to 200mM Tris-HCl).

In some embodiments, component i) comprises: 0.5-1.5% (v/v) Tween 20, 1-2% (v/v) Triton X-100, 1-2% (v/v) ethylphenyl polyethylene glycol, and 100-300 mol/L Na⁺And/or K⁺150mmol/L to 350 mol/L second strong base (such as sodium hydroxide and/or potassium hydroxide for example), 1% to 10% (w/v) adsorbent (such as Chelex resin for example) and aqueous solvent (such as one or more of water, buffer component and the like, preferably 50mM to 200mM Tris-HCl).

In some embodiments, component i) comprises: 0.7-1.3% (v/v) Tween 20, 1.2-1.8% (v/v) Triton X-100, 1.2-1.8% (v/v) ethylphenyl polyethylene glycol, 150-250 mol/L Na⁺And/or K⁺(for example, sodium salt and/or potassium salt, further example, sodium chloride and/or potassium chloride), 200mmol/L to 300mol/L second strong base (for example, sodium hydroxide or potassium hydroxide), 1% to 5% (w/v) adsorbent (for example, Chelex resin, etc.), and aqueous solvent (for example, one or more of water, buffer component, etc., preferably 50mM to 150mM Tris-HCl).

In one embodiment, component i) comprises: 1% (v/v) Tween 20, 1.5% (v/v) Triton X-100, 1.5% (v/v) ethylphenylpolyethylene glycol, 100mM sodium salt (exemplified independently by sodium chloride), 80mM potassium salt (exemplified independently by potassium chloride), 250mM strong base (exemplified by sodium hydroxide or potassium hydroxide), 1% to 5% (w/v) adsorbent (exemplified by Chelex resin and the like, in an amount of, for example, 1%, 2%, 3%, 4%, 5%), and water (such as sterilized purified water). Still further, 100mM Tris-HCl was included.

In some embodiments, component ii) comprises: 0.1 to 0.4mmol/L of a surfactant (e.g., sanskantin), 0.1 to 1.5% (w/v) of a dodecylbenzenesulfonate (e.g., SDS), 50 to 150mmol/L of a surfactant (e.g., sanskaton), and⁺and/or K⁺(preferably sodium salt)And/or potassium salt, further can be sodium chloride, potassium chloride), 0.2% -0.8% (v/v) ethanol, 250 mM-800 mM strong base (such as sodium hydroxide or potassium hydroxide for example), the solvent is water. In some embodiments, component ii) further comprises from 1% to 10% (w/v) of an adsorbent (e.g., Chelex resin, etc.).

In some embodiments, component ii) comprises: 0.1 to 0.4mmol/L of a surfactant (e.g., sanskantin), 0.3 to 1.2% (w/v) of dodecylbenzenesulfonate (e.g., SDS), 80 to 120mmol/L of K⁺(preferably sodium salt and/or potassium salt, further can be sodium chloride, potassium chloride), 0.3% -0.7% (v/v) ethanol, 250 mM-750 mM strong base (such as sodium hydroxide or potassium hydroxide for example) and 1% -5% (w/v) adsorbent (such as Chelex resin for example), further, the solvent is water.

In some embodiments, component ii) comprises: 0.25mmol/L surfactant (e.g. sanskantin), 1% (w/v) dodecylbenzenesulfonate (e.g. SDS), 100mmol/L K⁺(preferably sodium salt and/or potassium salt, further can be sodium chloride, potassium chloride), 0.5% (v/v) ethanol and 500mM strong base (such as sodium hydroxide or potassium hydroxide) and the like, for example, the use of 1%, 2%, 3%, 4%, 5%), solvent is water. Further, optionally, the adsorbent further comprises 1-5% (w/v) of an adsorbent (such as Chelex resin for example).

Optional preservation Components

In some embodiments, the viscous biological sample liquefaction release combination product further comprises a preservation component. The preservation component is used for stably preserving the liquefied sample and facilitating the subsequent operation of clinic in a flexible time.

The components of the preservation composition may be mixed to provide a separate preservative for preserving the viscous biological sample or the processed viscous biological sample.

The preservation component is a composition comprising the following components:

a) the buffer component (can be marked as a third buffer component) is used for adjusting the pH value of the preservation system to 6-8;

b) an osmotic pressure regulating component;

c) at least one of trehalose, mannitol, and glycerol.

The buffer component of the component a) can be used for neutralizing strong base in the liquefaction component and/or the nucleic acid release component, so that the pH environment of the liquefied sample is adjusted to a mild range, such as pH 6-pH 8, and a stable preservation environment is provided for the nucleic acid in the liquefied sample.

In some embodiments of the invention, component a) may adjust the pH of the preservation system to 6-8.

The buffer component of the preservative component, which is defined in accordance with the buffer component of the liquefaction component and the release component, serves to maintain a specific pH. It is to be understood that the definitions of the buffer component in the holding component, the liquefying component and the releasing component are independent of each other, may be the same as or different from each other, and may be independently selected from the "buffer components" defined in the present invention.

For the buffer components in the preservation component, buffers and buffer solutions are generally prepared from buffer salts or preferably non-ionic buffer components such as TRIS and HEPES, and may also be selected from weak acids and/or salts thereof. The buffer component used in i) is preferably selected from Tris-HCl of 0.5mmol/L to 500mmol/L, and the concentration thereof can be selected from 1mmol/L, 5mmol/L, 10mmol/L, 50mmol/L, 100mmol/L, 200mmol/L, 300mmol/L and 400 mmol/L. The buffer component in the preserving component that can be used in the method of the present invention is preferably selected from a) at least one of citric acid, acetic acid, phosphoric acid, tartaric acid, malic acid, carbonic acid, barbituric acid, or b) an acid radical of a, or c) an acid radical of a (usually carrying one or two hydrogen ions, e.g. hydrogen phosphate, dihydrogen phosphate), or one or more components selected from the group consisting of a), b), c).

The buffer component in the preservation component is used for neutralizing alkali, and after the sample is treated by the liquefied component and then mixed with the preservation component, the pH of the mixed system is approximately neutral, such as pH 6-pH 8, pH7 +/-0.2 and the like. In some embodiments of the invention, the concentration of the buffer component in the preservation component may be: 1mmol/L to 5mmol/L citric acid, and specific concentrations exemplified by 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, etc. may be selected. The concentration herein refers to the final concentration in the storage component, unless otherwise specified. The components of the buffer composition may be packaged separately in one or more containers. When the components are packaged separately in a container, it can be used as a pre-made buffer.

The osmolality adjusting component of the preservation component serves to stabilize the osmolality of the biological component (e.g., cells or viruses) to be preserved. The osmolality adjusting component generally comprises inorganic cations (in particular salt ions, preferably Na)⁺And K⁺) And/or betaine. In a specific embodiment, the osmolality adjustment component comprises 0.1% to 1.2% (w/v) sodium chloride and 0.1% to 1.2% (w/v) potassium chloride. In a specific embodiment, betaine is 0.1% to 10% (w/v). The concentration of the osmolality adjusting component, if not particularly limited, refers to the final concentration in the preservation component.

In some embodiments, the preservation component is provided in a single mixed system that can be formulated by adding other components to the cache component.

In some embodiments, the preservation component further comprises one or more amino acids. The amino acid can be a levorotatory or dextrorotatory chiral amino acid; can be natural amino acid or unnatural amino acid; examples of amino acids include, but are not limited to: such as glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, selenocysteine, pyrrolysine, and the like. In some preferred embodiments, the total concentration of amino acids in the storage component is preferably 1mol/L to 3mol/L, such as 1mol/L, 1.4mol/L, 1.5mol/L, 1.8mol/L, 2mol/L, 2.2mol/L, 2.5mol/L, 3 mol/L.

In some embodiments, component c) of the preserved component comprises 0.8mol/L to 1mol/L glycine and 0.6mol/L to 1mol/L isoleucine at a final concentration in the preserved component.

In some embodiments, the trehalose is at a concentration of 0.5mol/L to 1mol/L, e.g., 0.5mol/L, 0.6mol/L, 0.7 mol/L, 0.8mol/L, 0.9 mol/L, 1mol/L, etc., in the preservation component.

In some embodiments, the concentration of mannitol in the preservation component is 1.5% to 4.5% (w/v), and can also be 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, etc.

In some embodiments, the glycerol is present in the preservation component at a concentration of 2% to 10% (v/v), and can also be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.

In some embodiments, the storage component further comprises urea. The concentration of urea can be 1% to 3% (w/v), such as 1%, 1.5%, 2%, 2.5%, 3% (w/v), etc., based on the final concentration in the preservation component.

In some embodiments, the preservation component comprises (final concentration): 1 mmol/L-5 mmol/L citric acid, 0.1% -1.2% (w/v) sodium chloride, 0.1% -1.2% (w/v) potassium chloride, 0.8 mol/L-1 mol/L glycine, 0.6 mol/L-1 mol/L isoleucine, 0.5 mol/L-1 mol/L trehalose, 1.5% -4.5% (w/v) mannitol and 2% -10% (v/v) glycerol.

In some embodiments, the preservation component is provided as a single mixed system that can be formulated by adding other components to the buffer component. In some embodiments, the osmolality adjusting component is preferably 0.1% to 1.2% (w/v) sodium chloride, more preferably 1% (w/v), and the component c) of the preserving component is preferably 0.5mol/L to 1mol/L trehalose, and more preferably 1M trehalose.

The invention also relates to a kit comprising a combination product as described above.

If not specifically considered, the components of the liquefied component may be provided separately in different packaging containers or may be provided in combination in the same packaging container, the components of the released component may be provided separately in different packaging containers or may be provided in combination, and the components of the preserved component may be provided separately in different packaging containers or may be provided in combination. When combined, the mixture can be in liquid state, solid state or semi-solid state. The liquid state includes, but is not limited to, solution, emulsion, suspension, and the like. Examples of the semi-solid state include, but are not limited to, a gel state and the like.

In some embodiments, the components of the liquefaction component comprise an integrated, combined state.

In some embodiments, the components of the release component comprise an integrated, combined state.

In some embodiments, each of the components of the preserved component constitutes an integrated combined state.

In some embodiments, each of the liquefaction components, some components are present in combination with other components, and some components are present independently. In some embodiments, the rigid microparticles are present independently.

In some embodiments, the buffer component in the liquefaction component is present independently.

In some embodiments, the buffer component of the release component is present independently.

In some embodiments, the buffer component of the preservation component is present independently.

In some embodiments, the buffer components in the liquefaction component, the release component, and the preservation component are provided by buffer reagents of the same component class. Further, the contents of the components contained are also the same.

In some embodiments, each of the stored components, some components and other components are present in a component state, and some components are present independently. In some preferred embodiments, component a), component b), and component c) are each independently present. In some preferred embodiments, component b) and component a) are present in combination, and component c) is also present in combination with component a).

In some embodiments, the kit further comprises one or more of an extraction agent, an amplification agent, and a detection agent for the nucleic acid.

In some embodiments, the nucleic acid comprises DNA and/or RNA, which may be in a variety of forms, and may be in the form of short oligonucleotides, long oligonucleotides, or polynucleotides. Double-stranded DNA and single-stranded DNA, and double-stranded RNA and single-stranded RNA are also possible. In some embodiments, the nucleic acid can be a gene, a cDNA molecule, an mRNA, a tRNA, an rRNA, a non-coding RNA molecule, and the like, as well as fragments of the above nucleic acid forms, such as oligonucleotides.

According to still another aspect of the present invention, there is also provided a method for liquefying and releasing a viscous biological specimen, comprising the steps of:

2) incubating the first mixture to obtain a second mixture;

3) the second mixture is mixed with the nucleic acid releasing component as described above and releases the nucleic acid in the sample.

The step 1) is to provide a suitable pH environment for the subsequent liquefaction.

The pH of the system at which liquefaction is carried out is preferably basic, and the basicity is preferably sufficient to provide a strong alkaline environment at a pH of 10 or more.

In some embodiments, guaifenesin and the first strong base can be packaged separately, and at this time, the addition amount of the first strong base can be flexibly controlled according to the characteristics of the viscous biological sample, so that the pH value of the liquefaction system can be flexibly controlled, and the differentiation characteristics among different samples can be better adapted.

In some embodiments, the guaifenesin and the strong base can be packaged separately, and at the moment, the addition amount of the strong base can be flexibly controlled according to the characteristics of the viscous biological sample, so that the pH value of a liquefaction system can be flexibly controlled, and the method can better adapt to the differentiation characteristics among different samples.

Likewise, in some embodiments, the content of guaifenesin can be flexibly controlled, so that the liquefaction process of a trace sample can be more accurately controlled.

In some embodiments, the first mixture has a pH of 10 or more, such as pH10, pH11, pH11.5, pH12, pH12.5, pH13, pH13.5, pH14, pH14.5, pH15, preferably pH12 to pH14. Adjusting the pH of the first mixture may control the pH of the ii) stepwise incubation.

In some embodiments, the concentration of guaifenesin in the first mixture (i.e., the working concentration of guaifenesin) is from 1mmol/L to 1mol/L, e.g., 1mmol/L, 5mmol/L, 10mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L, 100mmol/L, 110mmol/L, 120mmol/L, 130mmol/L, 140mmol/L, 150mmol/L, 200mmol/L, 300mmol/L, 400mmol/L, 500mmol/L, 600mmol/L, 700mmol/L, 800mmol/L, 900mmol/L, 1mol/L, and the like.

The step 2) is to liquefy and reduce the viscosity of the sample.

In the present invention, the liquefaction is carried out without requiring severe temperature conditions, for example, at room temperature.

In some embodiments, the incubation temperature is between 18 ℃ and 35 ℃. Examples of incubation temperatures include, but are not limited to: 18 ℃, 19 ℃, 20 ℃,21 ℃,22 ℃,23 ℃,24 ℃, 25 ℃,26 ℃, 27 ℃,28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃ and the like.

In some embodiments, the incubation time is ≧ 5 min. Examples of incubation times include, but are not limited to, 5min, 6min, 7min, 8min, 9min, 10min, 15min, 20min, 30min, or more.

When liquefaction is carried out, the incubation time can be reasonably controlled according to factors such as the sample amount, the sample characteristics and the like, and sufficient liquefaction is realized in a reasonable time.

In some embodiments, from the clinical operability, 5min ~30min is relatively preferred.

In some embodiments, step 2) further comprises: the incubated first mixture is mixed with preservation components as described above for preservation. That is, after liquefying the viscous biological sample, the viscous biological sample is preserved with a preservation component, and then subjected to nucleic acid release treatment when necessary.

In some embodiments, the volume ratio of the preservation component to the first mixture is 1: (2-4), 1:2, 1:2.5, 1:3, 1:3.5, 1:4, etc. may be selected.

The above step 3) is to effect the release of the nucleic acid.

In some embodiments, the nucleic acid releasing component is a composition as set forth in component ii) and is effective to release DNA.

In some embodiments, the nucleic acid releasing component is a composition according to component i) and is effective to release DNA and also effective to release RNA.

In some embodiments, the nucleic acid releasing component is a composition comprising component i) and component ii) and is effective to release both DNA and RNA.

In some embodiments of the invention, the nucleic acid releasing component single mixed system is provided in a single package.

In some embodiments, the volume ratio of the second mixture to the nucleic acid releasing component in step 3) is 1: (0.5 to 1.5).

The mixing method in step 1) is not particularly limited as long as the sample is sufficiently contacted with the liquefied component and the liquefaction can be smoothly performed.

The mixing method in step 3) is also not particularly limited as long as the release of nucleic acid can be smoothly achieved.

In some embodiments, in step 1) and/or 3), the fluids are mixed with stirring or shaking.

In some embodiments, the viscous biological sample is selected from the group consisting of sputum and cervical mucus.

In some embodiments, the viscous biological sample is sputum and/or cervical mucus.

According to still another aspect of the present invention, there is also provided a method for extracting nucleic acid from a viscous biological sample, comprising the steps of:

extracting the released nucleic acids using a nucleic acid extracting agent.

The term "extraction of nucleic acids" refers to a process of separating the released nucleic acids from impurities, often accompanied by purification of the nucleic acid components. Such as centrifugation, enrichment of magnetic beads, stratification of sample components by chemical reagent treatment, etc.

According to still another aspect of the present invention, there is also provided a method for amplifying a nucleic acid in a viscous biological sample, comprising the steps of:

optionally extracting the released nucleic acids using a nucleic acid extracting agent;

amplifying the nucleic acid using a nucleic acid amplification agent.

The term "amplification" when co-occurring in the context of the term "nucleic acid" refers to the production of multiple copies of a polynucleotide, or portion of a polynucleotide, usually starting from a small amount of the polynucleotide (e.g., as little as a single polynucleotide molecule), wherein the amplification product or amplicon is usually detectable. Amplification of polynucleotides includes a variety of chemical and enzymatic methods. The generation of multiple copies of DNA from one or several copies of a target or template DNA molecule during Polymerase Chain Reaction (PCR), Rolling Circle Amplification (RCA) or Ligase Chain Reaction (LCR) is a form of amplification. Amplification is not limited to the strict replication of the starting molecule. For example, the use of reverse transcription RT-PCR to generate multiple cDNA molecules from a limited amount of RNA in a sample is an amplified version. In addition, the production of multiple RNA molecules from a single DNA molecule during the transcription process is also an amplified version.

According to still another aspect of the present invention, there is also provided a method for detecting nucleic acid in a viscous biological sample, comprising the steps of:

optionally amplifying the nucleic acid using a nucleic acid amplification agent;

detecting the nucleic acid using a nucleic acid detecting agent.

Methods of detecting nucleic acids may or may not rely on amplification of the nucleic acid. As used herein, the term "method of detection of a nucleic acid" refers to any method of determining the nucleotide composition of a nucleic acid of interest, including, but not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays, (Hologic, Inc.) and described, for example, in U.S. Pat. Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543 and 6,872,816; Lyamichev et al, nat. Biotech, 17:292(1999), Hall et al, PNAS, USA,97:8272(2000), and US 2009/0253142, each of which is incorporated herein by reference in its entirety for all purposes); enzymatic mismatch cleavage methods (e.g., variaginics, U.S. patent nos. 6,110,684, 5,958,692, 5,851,770, which are incorporated herein by reference in their entirety); the Polymerase Chain Reaction (PCR) described above; branched hybridization methods (e.g., Chiron, U.S. Pat. nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, which are incorporated herein by reference in their entirety); rolling circle replication (e.g., U.S. Pat. nos. 6,210,884, 6,183,960, and 6,235,502, which are incorporated herein by reference in their entirety); NASBA (e.g., U.S. patent No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated herein by reference in its entirety); e-sensor technology (Motorola, U.S. patent nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated herein by reference in their entirety); circular probe technology (e.g., U.S. Pat. nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated herein by reference in their entirety); dade Behring signal amplification methods (e.g., U.S. Pat. nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated herein by reference in their entirety); ligase chain reaction (e.g., Baranay proc. natl. acad. sci USA 88,189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, which is incorporated herein by reference in its entirety).

The invention also relates to the application of the viscous biological sample liquefaction release combination product and the kit thereof in the direct amplification or direct detection of nucleic acid.

In some embodiments of the invention, the use is for non-diagnostic and therapeutic purposes.

In some embodiments of the invention, the use is for diagnostic and therapeutic purposes.

Embodiments of the present invention will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the conditions not specified in the following examples, preferably with reference to the guidelines given in the present invention, may also be performed according to the experimental manual or the conventional conditions in the art, and may also be performed according to other experimental procedures known in the art, or according to the conditions suggested by the manufacturer.

In the following specific examples, the measurement parameters relating to the components of the raw materials, if not specified otherwise, may be subject to slight deviations within the accuracy of the weighing. Temperature and time parameters are involved to allow for acceptable variation in instrument test accuracy and/or operational accuracy.

The concentration unit of DTT and dithiothreitol is mass-to-volume ratio.

SDS, sodium dodecyl sulfate.

Nucleic acid extraction or purification reagents (RNA one-step release reagents) produced by saint xiang biotechnology, ltd, used in the examples, specification types: specification 24T, model S1014.

The six respiratory pathogen nucleic acid detection kits (PCR-fluorescent probe method) used in the examples were 2021001 batches, national Standard 20213400256.

Example 1 liquefaction Effect test

In this example, the liquefaction reagent of the experimental group was prepared from guaifenesin, sodium hydroxide, and zirconia beads. The solvent used for preparing the reagent was purified water at a final concentration of 100mM guaifenesin, 10mM sodium hydroxide and 1g/mL zirconia beads (1 mM in diameter), and this was designated as an experimental group.

The 4 visually viscous sputum samples (I, II, III, IV) were collected, mixed well individually and 6 samples of 2mL were taken, each numbered 1-6. And (5) standby.

Adding 6mL of experimental group reagent into the sample No. 1;

to sample No. 2 was added 6mL of dithiothreitol solution ((2% (w/v) DTT);

to sample No. 3 was added 6mL of sodium hydroxide solution (0.1M);

adding 6mL of reagent prepared by 100mM acetylcysteine, 10mM sodium hydroxide and 1g/mL of zirconia beads (the diameter of a through hole is 1 mM) into the sample No. 4;

to sample No. 5 was added 6mL of guaifenesin at a final concentration of 100 mM;

10mM sodium hydroxide is added to sample No. 6, the mixture is shaken and mixed for 5 minutes at room temperature to process the sputum, the pH value is adjusted to be neutral, and then 100mM guaifenesin is added, and the mixture is shaken and mixed for 5 minutes at room temperature.

The samples are uniformly shaken and mixed for 5 minutes and are carried out at room temperature;

the liquefied 4 samples obtained in the above step were each aspirated by a pipette, and the aspiration process was observed and recorded, with the results shown in table 1.

The result shows that the sputum liquefied by the method can be smoothly sucked by a 10 mu L pipette, and the result proves that the method has good effect of liquefying the sputum and obviously reduces the viscosity of the sample.

The use of acetylcysteine (sample No. 4) instead of guaifenesin in the present system was found to have a reduced liquefaction effect. In addition, in the case of guaifenesin alone (sample No. 5), the liquefaction effect also decreased.

Table 1.

In addition, in the single guaifenesin group (No. 5, no zirconia beads are added for treatment for 30min under normal temperature neutral condition), the liquefaction effect is general, and the individual thick sputum suction heads have the phenomenon of wire drawing when sucking, but no sputum is visible. For sample No. 5, the liquefaction effect of the sample can be basically equivalent to that of the experimental group under the condition of heating at 95 ℃ for 30 min.

Example 2 Effect test of different concentrations of liquefaction Components on zirconia beads

In this example, the test group liquefaction reagent consists of one or more of the following components; guaifenesin, sodium hydroxide, zirconia beads.

Experimental group 1: the final concentration was 100mM guaifenesin, 10mM sodium hydroxide, and 1g/mL zirconia beads (1 mM in diameter), and the solvent used for preparing the reagent was purified water.

Experimental group 2: the final concentration was 1mM guaifenesin, 1000mM sodium hydroxide, and 1g/mL zirconia beads (1 mM in diameter), and the solvent used for preparing the reagent was purified water.

Experimental group 3: the solvent used for preparing the reagent was purified water at a final concentration of 1mM guaifenesin, 1g/mL zirconia beads (1 mM in diameter) and 0.1mM sodium hydroxide.

The 4 visually viscous sputum samples (I, II, III, IV) were collected, mixed well individually for each and 3 samples of 2mL, numbered 1-3, were taken for each. And (5) standby.

To sample No. 1, add 6mL of experimental group 1 reagent (pH =12 after mixing);

to sample No. 2, add 6mL of experimental group 2 reagent (pH =14 after mixing);

add 6mL of experimental group 3 reagent to sample No. 3 (pH =10 after mixing);

the liquefied 4 samples obtained in the above step were each aspirated by a pipette, and the aspiration process was observed and recorded, with the results shown in table 2.

The results show that the sputum liquefied by the methods of each experimental group can be smoothly sucked by a 10 mu L pipette, and the results prove that the method has good liquefaction effect on the sputum under wider guaifenesin concentration and pH, and the sample viscosity is obviously reduced.

Table 2.

Example 3 Rapid detection and comparison with other methods for liquefied sputum samples

In this example, the liquefaction reagent of the experimental group was prepared from guaifenesin, sodium hydroxide, and zirconia beads. The reagent was prepared in the form of purified water at a final concentration of 100mM guaifenesin, 10mM sodium hydroxide, and 1g/mL zirconia beads (1 mM diameter) and used as a test group.

The 4 visually viscous sputum samples (I, II, III, IV) were collected, mixed well individually, and 5 2mL samples were taken, numbered 1-5, for each sample. And (4) standby.

Adding 6mL of experimental group reagent into the sample No. 1; to sample No. 2, 2.5g of guanidine hydrochloride + 0.5 g of acetylcysteine +2g of polypropylene particles (method disclosed in patent document No. CN 108949748A); add 6mL dithiothreitol solution (2% DTT) to sample No. 3; to sample No. 4 was added 6mL of trypsin solution (buffer system containing pH7.0); to sample No. 5, 6mL of sodium hydroxide solution (0.1M) was added, and the above samples were shaken and mixed for 5 minutes at room temperature.

The liquefied 4 samples obtained in the above step were each aspirated by a pipette (non-liquefied sample was centrifuged at 12000rmp for 1min to obtain the supernatant).

Nucleic acid extraction or purification reagents (RNA one-step release agents) produced by Shengxiang biological science and technology Co., Ltd are adopted for extraction, and six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescent probe method) produced by Shengxiang biological Co., Ltd are adopted for detection. The detection results are shown in table 3, and the Ct values of the respiratory pathogen nucleic acid detection of different samples in this example are listed in table 3; wherein the Ct value represents the cycle number of the fluorescence signal in each reaction tube reaching a set threshold, and the Ct value of each template has a linear relation with the logarithm of the initial copy number of the template, and the formula is as follows.

X₀For initial template amount, Ex is amplification efficiency,Nis the amount of amplified product when the fluorescent amplification signal reaches a threshold intensity. n is the number of cycles of the amplification reaction, and n = Ct when the amplification reaches the threshold line.

The higher the initial copy number, the lower the Ct value. A standard curve can be made using a standard with a known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Therefore, once the Ct value of an unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.

Therefore, the value of Ct can reflect the concentration of the detected sample, the lower the Ct value is, the higher the nucleic acid concentration of the detected sample is, and the higher the Ct value is, the lower the nucleic acid concentration of the detected sample is; samples that differ by 1 Ct value have a two-fold difference in nucleic acid concentration, and differ by 2 Ct values by four-fold difference, and so on. According to Table 3, after 4 liquefied samples of the experimental group of this example were tested, human genomic DNA was detected normally (Ct values were all less than 40). The respiratory syncytial viruses are all positive (Ct values are all less than 40), and the Ct values of DNA and RNA detection by other methods are influenced by different degrees. The other methods can not directly carry out rapid extraction and detection after liquefying the sample. There is resistance to POCT (point of care) detection systems. The amplification curves of the above experimental results are shown in FIG. 1 and FIG. 2, respectively. In FIG. 1, A is the experimental group; b is a protease method; c is a sodium hydroxide method; d is DTT method; e is an acetylcysteine method. In FIG. 2, A is an experimental group; b is a protease method; c is a sodium hydroxide method; d is DTT method; e is an acetylcysteine method. The ordinate Rn in FIGS. 1 and 2 represents the fluorescence intensity of the PCR amplification product at the nth cycle. The results show that the groups A, B and D can be amplified normally, which indicates that the 3 groups all meet the requirements of the reagent nucleic acid amplification detection. The highest concentration of the Ct values in the group A indicates that the sample liquefied in the method has the highest efficiency of detecting the target nucleic acid fragment.

TABLE 3 Ct values for the detection of nucleic acids of respiratory pathogens in different samples of example 3.

Example 4 validation of the Effect of the extraction-free amplification System (nucleic acids extracted by introduction of amplification were not purified)

In this example, the liquefaction reagent of the experimental group was prepared from guaifenesin, sodium hydroxide, and zirconia beads. The solvent used for preparing the reagent was purified water at a final concentration of 100mM guaifenesin, 10mM sodium hydroxide, and 1g/mL zirconia beads (1 mM in diameter).

2 parts (sample No. 1 and sample No. 2) of throat swab samples were selected, diluted 100 times with the liquefaction reagent and normal saline (control group) of the experimental group of this example as the substrate, and then extracted with the nucleic acid extraction or purification reagent (RNA one-step release agent) produced by SANXIANG Biotechnology GmbH, and detected with the nucleic acid detection kit (PCR-fluorescent probe method) for six respiratory pathogens produced by SANXIANG Biotech. The results are shown in Table 4 (Ct value) and FIG. 3. The results show that, after the experiment group of this example dilutes 2 selected samples, the concentration value of the nucleic acid tested by using the fluorescence quantitative PCR method extraction-free amplification system is very small from the concentration value of the nucleic acid tested by diluting the physiological saline substrate, which indicates that the reagents in the experiment group of this example have no inhibition effect on the extraction of the rear-end nucleic acid and the amplification detection by the fluorescence quantitative PCR method. Therefore, the application friendliness to the back end is strong. As can be seen from FIG. 3, both matrix-diluted test nucleic acid curve shapes were amplified normally with a small difference in Ct values.

TABLE 4 Ct values for nucleic acid detection of respiratory pathogens in different samples of example 4

Example 5 sample stability testing after liquefaction.

In this example, the liquefaction reagent of the experimental group was prepared from guaifenesin, sodium hydroxide, and zirconia beads. The final concentration was 100mM guaifenesin, 10mM sodium hydroxide, and 1g/mL zirconia beads (1 mM in diameter), and the solvent used for preparing the reagent was purified water.

1 sample of visually more viscous sputum was collected and divided into 2 portions. One part of the extract is added into a 3-volume physiological saline grinder to be ground and homogenized for later use. The other sputum sample was mixed with the liquefaction reagent of the experimental group of this example, and was divided into 11 samples, numbered 1-11. And (5) standby. The liquefied sample is placed at 37 ℃ for 0h, 4h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h and 192h, and is respectively extracted by nucleic acid extraction or purification reagents (RNA one-step release agents) produced by Santa Clan Biotechnology Ltd, and six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescent probe method) produced by Santa Clan Biotechnology Ltd. The detection results are shown in table 5 (Ct values), and the results show that the samples subjected to liquefaction treatment in the experimental group of the present embodiment can be stably detected within 24 hours by using a rapid purification-free detection system to detect RNA, and the concentration does not change significantly within 4 hours. In the embodiment, DNA of the sample subjected to liquefaction treatment in the experimental group can be stably detected within 192h by adopting a rapid purification-free detection system, and the concentration does not change obviously within 144 h. RNA and DNA were detected at 37 ℃ for nucleic acid comparable to the control. The results of the experiment are shown in table 5 and fig. 4. As can be seen from FIG. 4, all 4 pictures were amplified normally, and the curve shape of A in FIG. 4 is consistent with that of C in FIG. 4. The shape of B in FIG. 4 is more consistent with that of C in FIG. 4. The difference of the storage time of the nucleic acid of the experimental group and the control group is small.

TABLE 5 Ct values for nucleic acid detection of respiratory pathogens in different samples of example 5

Example 6 Effect test on liquefying cervical mucus

In this example, the liquefaction reagent of the experimental group was prepared from guaifenesin, sodium hydroxide, and zirconia beads. The final concentration was 100mM guaifenesin, 10mM sodium hydroxide, and 1g/mL zirconia beads (diameter: 1 mM), and the solvent used for preparing the reagent was purified water.

4 visually-measured and relatively viscous cervical mucus samples collected in the Saint Valer medical testing center are fully mixed with the liquefaction reagent of the experimental group of the embodiment according to the volume ratio of 1:3, and are uniformly mixed for 5 minutes by oscillation.

The liquid transfer machines are used for respectively sucking the liquefied 4 samples obtained in the previous step, the sucking process is observed and recorded, and the result is shown in table 6.

Table 6.

Example 7 comparative testing of the ability of the preservative fluid of the invention to preserve liquefied viral samples and nucleic acids with commercial preservative fluids

2 clinical sputum samples (sample No. 1 and sample No. 2) were taken, which contained respiratory viruses. The liquefaction was carried out using the method of the experimental group in example 1. After liquefaction was complete, the preservative solution and commercial preservative solution provided in the experimental group of this example were added.

The experimental group of this example was prepared with a preservative solution reagent: the concentrations of the following components are final concentrations after preparation, namely 3mM of citric acid, 1% (w/v) of sodium chloride, 0.8% (w/v) of potassium chloride, 1M of trehalose, 2.5% (w/v) of mannitol, 1.5% (w/v) of urea, 6% (v/v) of glycerol, 1M of glycine and 1M of isoleucine, and the solvent is sterilized purified water.

The storage solution 1/3 times the volume of the sample is added to the sample after complete liquefaction and mixed well.

Commercial preservation solution (the main active ingredients include RNase, guanidine thiocyanate and sodium chloride, and the components of the preservation solution are greatly different from those of the experimental group in the embodiment): operating according to the instructions.

Samples preserved by the two preservation solutions are respectively placed for 0h, 4h, 8h, 24h, 48h, 72h, 96h, 144h, 192h and 288h at 37 ℃ and are synchronized (the samples are placed at-20 ℃ for preservation after being treated at 37 ℃ and are unfrozen for standby after all test groups are completely treated), and nucleic acid extraction or purification reagents (RNA one-step release agents) produced by Santa Clan Biotechnology Ltd and six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescent probe method) produced by the Santa Clan biological company are respectively adopted for extraction.

The results of the detection are shown in Table 7 (Ct value) and FIG. 5, which show that the storage solution of the experimental group of this example stored the sputum sample liquefied by the liquefaction method, and that the detectable time of the viral nucleic acid was longer than that of the commercial storage solution, and the detection concentration was higher than the concentration value of the commercial storage solution. From the change of the nucleic acid concentration in the period of 0h-288h, the nucleic acid degradation degree of the storage solution of the experimental group is obviously lower than that of the commercial storage solution, which shows that the storage solution of the experimental group has great advantages, better stability and longer durability when the virus sample and the nucleic acid are stored by using fluorescence PCR detection.

TABLE 7 Ct values of the results of the detection of the nucleic acid of the respiratory virus in the different samples of example 7

Example 8 comparative testing of the ability of the preservative fluid of the invention to preserve pathogen samples and nucleic acids after liquefaction with commercial preservative fluids

2 clinical sputum samples (sample No. 1 and sample No. 2) were selected, which contained gonococci. The liquefaction was carried out using the method of the experimental group in example 1. After liquefaction was complete, the preservative solution and commercial preservative solution provided in the experimental group of this example were added.

Commercial preservation solution (main active ingredients include RNasin, guanidine thiocyanate, sodium chloride): operating according to the instructions.

The samples preserved by the two preservation solutions are respectively placed for 0h, 4h, 8h, 24h, 48h, 72h, 96h, 144h, 192h and 288h at 37 ℃ and are synchronized (the samples are placed at-20 ℃ for preservation after being treated at 37 ℃ and are unfrozen for standby after all the test groups are completely treated), and nucleic acid extraction or purification reagents (RNA one-step release agents) produced by Santa Hunan Biotechnology corporation are respectively adopted for extraction, and a gonococcus nucleic acid detection kit (PCR-fluorescent probe method) produced by the Santa Hunan Biotechnology corporation is adopted.

The results of the tests are shown in Table 8 (Ct value) and FIG. 6, and show that the specimen of the sputum liquefied by the liquefaction method stored in the storage solution of the experimental group of this example has a detectable time of pathogenic bacteria nucleic acid substantially equivalent to that of the commercial storage solution, and has a higher detection concentration than that of the commercial storage solution. The change of the nucleic acid concentration in the period of 0h-288h can be found that the degradation degree of the nucleic acid of the preservation solution for the experimental group is obviously lower than that of the commercial preservation solution, which indicates that the preservation solution for the experimental group has a certain advantage in the preservation of the virus sample and the nucleic acid by using the fluorescent PCR detection.

TABLE 8 Ct values of gonococcal DNA nucleic acid detection results of different samples in example 8

Example 9 comparative testing of the ability of the preservation solution of the invention to preserve liquefied cell samples and nucleic acids with a commercial preservation solution

2 clinical sputum samples (sample No. 1 and sample No. 2) were selected, and the samples contained human exfoliated cells and the like. The liquefaction was carried out using the method of the experimental group in example 1. After liquefaction was complete, the preservative solution and commercial preservative solution provided in the experimental group of this example were added.

The preparation of the preservation solution reagent in the experimental group embodiment: the concentrations of the following components are final concentrations after preparation, namely 3mM of citric acid, 1% (w/v) of sodium chloride, 0.8% (w/v) of potassium chloride, 1M of trehalose, 2.5% (w/v) of mannitol, 1.5% (w/v) of urea, 6% (v/v) of glycerol, 1M of glycine and 1M of isoleucine, and the solvent is sterilized purified water.

The results of the tests are shown in Table 9 (Ct values) and FIG. 7, which show that the storage solution of this example stored the sputum sample liquefied by the liquefaction method, and that the human genome nucleic acid was detectable in a time comparable to that of the commercial storage solution, and in a concentration higher than that of the commercial storage solution. The change of the nucleic acid concentration in 0h-288h shows that the degradation degree of the nucleic acid of the preservation solution is obviously lower than that of the commercial preservation solution, which shows that the preservation solution has certain advantages when the fluorescent PCR is used for detecting and preserving the virus sample and the nucleic acid.

TABLE 9 Ct values of nucleic acid detection results of human genomic DNA of different samples in example 9

Example 10 comparative testing of nucleic acid releasing Agents of the invention with commercially available nucleic acid releasing Agents for the ability to release nucleic acid from liquefied sputum samples

10.1. Formulation 10-1

The experimental groups of this example were formulated with release agents: the following component concentrations are final concentrations after preparation, the volume percentage of Tween 20 is 1% (v/v), the volume percentage of Triton X-100 is 1.5% (v/v), the volume percentage of ethyl phenyl polyethylene glycol is 1.5% (v/v), the mass concentration of strong base (specifically sodium hydroxide) is 10mg/mL (250 mM), the mass volume percentage of Chelex resin is 5% (w/v), and the solvent is sterilized purified water.

Commercial nucleic acid releasing agent 1: the main components comprise guanidinium isothiocyanate, Sodium Dodecyl Sulfate (SDS) and ethanol.

Commercial nucleic acid releasing agent 2: the main components comprise sodium hydroxide, potassium chloride, isoamylol and tween.

2 clinical sputum samples (sample No. 1 and sample No. 2) were selected, which contained respiratory viruses, human cells, and gonococci. The liquefaction was carried out using the method of the experimental group in example 1. After the sample is completely liquefied, the experimental group nucleic acid releasing agent and the commercial nucleic acid releasing agent are added for cracking treatment according to requirements. After the sample is completely processed, six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescence probe method) and gonococcal nucleic acid detection kits (PCR-fluorescence probe method) produced by Shengxiang biological science and technology Co.

Experimental groups of this example nucleic acid releasing agents: and (3) taking 10 mu L of the sample completely liquefied and 10 mu L of the nucleic acid releasing agent, fully and uniformly mixing, and standing for 10min at room temperature. The nucleic acid is ready for use.

Commercial nucleic acid releasing agent 1: the procedure was followed (15. mu.L of sample and 5. mu.L of nucleic acid releasing agent, pipette tip was repeatedly and thoroughly mixed, and the mixture was allowed to stand at room temperature for 10 min). The nucleic acid is ready for use.

Commercial nucleic acid releasing agent 2: the procedure was followed (sampling 25. mu.L of the sample with 25. mu.L of the nucleic acid releasing agent, mixing the tip by repeated pipetting, and standing at room temperature for 10 min). The nucleic acid is ready for use.

Two samples No. 1 and No. 2 were set for each set.

The detection results (Ct values) are shown in tables 10 to 12 below and FIG. 8, in FIG. 8, A is an RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a No. 2 sample human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is a sample No. 2 gonococcus DNA amplification curve; the Ct position (the position of the intersection of the horizontal line and the amplification curve in the figure) corresponding to the reagent of the present application is also shown in FIG. 8. In tables 10-12, the Ct value of the release agent of the present application is at least less than 1 compared to the commercial release agent, i.e., the sensitivity is significantly improved. Particularly, in table 10, when respiratory viruses are detected, the Ct values are 8.13 and 9.74 lower than those of commercial reagents, and the sample concentration is reflected by the Ct values, which shows that the efficiency of the reagent of the present application on the release of sample nucleic acid is significantly improved compared with commercial reagents. The results show that the concentration of the nucleic acid releasing agent detecting virus in the experimental group of this example is higher than that of the commercial releasing agent. It is demonstrated that the releasing agent provided in this example has great advantages in the detection of RNA virus sample nucleic acid using fluorescence PCR. In this example, the concentration of DNA detected by the nucleic acid releasing agent was higher than that detected by the commercial releasing agent. It is demonstrated that the release agent of the experimental group of the present embodiment also has certain advantages in detecting DNA sample nucleic acid by using fluorescence PCR.

Table 10.

Table 11.

Table 12.

10.2. Formulation 10-2, formulation 10-3 and formulation 10-4

According to the following release reagent formula with different component dosage, a new sputum sample containing respiratory viruses, gonococci and human cells is tested by adopting the method of part 10.1. The results show that the released reagent components of the application treat samples in different concentration ranges, and the fluorescence PCR detection is used after nucleic acid is obtained, so that the released reagent components have higher sensitivity.

Formula 10-2: 0.1% (v/v) Tween-20, 1.5% (v/v) Triton X-100, 0.1% (v/v) ethylphenylpolyethylene glycol, 20mmol/L sodium chloride, 50mmol/L sodium hydroxide, 1% (w/v) Chelex resin and sterilized purified water. The results are shown in table 13 and fig. 12.

TABLE 13 formulation 10-2 test results

And (3) formula 10-3: 1% (v/v) Tween-20, 3% (v/v) Triton X-100, 1% (v/v) ethylphenylpolyethylene glycol, 200mmol/L sodium chloride, 500mmol/L sodium hydroxide, 5% (w/v) Chelex resin and sterilized purified water. The results are shown in table 14 and fig. 13.

TABLE 14 formulation 10-3 test results

And (3) formula 10-4: 2% (v/v) Tween-20, 0.1% (v/v) Triton X-100, 3% (v/v) ethylphenylpolyethylene glycol, 1000 mmol/L sodium chloride, 1250 mmol/L sodium hydroxide, 15% (w/v) by mass volume of Chelex resin, and sterilized purified water. The results are shown in table 15 and fig. 14.

TABLE 15 test results for formulations 10-43

Example 11 comparative testing of the ability of nucleic acid releasing agents of the invention to treat sputum samples which have been liquefied and added to a preservation solution

The experimental groups of this example were formulated with release agents: the concentrations of the following components are final concentrations after preparation, the molar concentration of Tris-HCl is 100mmol/L, the volume percentage of Tween 20 is 1% (v/v), the volume percentage of Triton X-100 is 1.5% (v/v), the volume percentage of ethyl phenyl polyethylene glycol is 1.5% (v/v), the mass concentration of strong base and strong base (specifically sodium hydroxide) is 10mg/mL (250 mM), the mass volume percentage of Chelex resin is 5% (w/v), and the solvent is sterilized purified water.

Commercial nucleic acid releasing agent 1 and commercial nucleic acid releasing agent 2 were the same as in example 10.

2 clinical sputum samples were selected, which contained respiratory viruses, human cells, and gonococci. Liquefaction and storage were performed by the method of the experimental group in example 7. After the samples are stored for 2 days, the samples are respectively added with the nucleic acid releasing agent and the commercial nucleic acid releasing agent of the experimental group for cracking treatment according to requirements. After the sample was completely processed, six respiratory pathogen nucleic acid detection kits (PCR-fluorescent probe method) and gonococcus nucleic acid detection kits (PCR-fluorescent probe method) manufactured by Shengxiang Biotechnology corporation were used for detection.

Experimental groups of this example nucleic acid releasing agents: and (3) taking 10 mu L of the sample completely liquefied and 10 mu L of the nucleic acid releasing agent, fully and uniformly mixing, and standing for 10min at room temperature.

Commercial nucleic acid releasing agent 1: the procedure was followed (15. mu.L of sample and 5. mu.L of nucleic acid releasing agent, pipette tip was repeatedly pipetted and mixed well, and left at room temperature for 10 min). The nucleic acid is ready for use.

The detection results (Ct values) are shown in tables 16 to 18 below and FIG. 9, in FIG. 9, A is an RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a No. 2 sample human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is the sample No. 2 gonococcus DNA amplification curve. The results show that the nucleic acid concentration of the RNA viral samples with the preservation solution matrix treated by the nucleic acid releasing agent provided in this example was higher than the concentration of the RNA in the treated samples treated by the commercial releasing agent when the RNA viral samples were detected by using six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescent probe method) manufactured by Santa Xiang Biotechnology Ltd. It is demonstrated that the test panel of this example shows a higher efficiency of treatment of the R NA virus sample containing the preservative fluid matrix with the release agent in the case of nucleic acid detection using fluorescence PCR methodology.

After treating a DNA sample with a preservation solution matrix with the nucleic acid releasing agent provided in this example, six respiratory pathogen nucleic acid detection kits (PCR-fluorescent probe method) manufactured by Santa Clan Biotechnology Ltd were used for detection, and the concentration of DNA nucleic acid was higher than that of a commercial releasing agent. It is demonstrated that the test panel of this example also shows a higher efficiency of treatment of the DNA sample containing the preservative fluid matrix with the release agent in the case of nucleic acid detection using fluorescence PCR methodology.

Table 16.

Table 17.

Table 18.

Example 12 comparative testing of nucleic acid Release Agents of the invention with commercially available nucleic acid Release Agents for the Release of liquefied sputum sample nucleic acid

12.1. Formulation 12-1

The experimental groups of this example were formulated with release agents: the following component concentrations are prepared final concentration, 100mmol/L potassium chloride, 0.25mmol/L cypersantine, 1% sodium dodecyl benzene sulfonate by mass volume ratio, 0.5% (v/v) ethanol, 500mmol strong base (sodium hydroxide), and sterilized purified water as solvent.

2 clinical sputum samples (sample No. 1 and sample No. 2) were selected, which contained respiratory viruses, gonococci, and human cells. The liquefaction was carried out using the method of the experimental group in example 1. After the sample is completely liquefied, the nucleic acid releasing agent and the commercial nucleic acid releasing agent are added according to requirements for cracking treatment. After the sample is completely processed, six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescence probe method) and gonococcal nucleic acid detection kits (PCR-fluorescence probe method) produced by Shengxiang biological science and technology Co.

The detection results (Ct values) are shown in tables 19 to 21 and FIG. 10, in FIG. 10, A is the RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a No. 2 sample human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is the sample No. 2 gonococcus DNA amplification curve. The results show that the concentration of the nucleic acid releasing agent detecting virus in the experimental group of this example is higher than that of the commercial releasing agent. The releasing agent has certain advantages when the releasing agent is used for detecting RNA virus sample nucleic acid by using fluorescence PCR. In this example, the concentration of DNA detected by the nucleic acid releasing agent was higher than that detected by the commercial releasing agent. It is demonstrated that the release agent of the experimental group in this example has great advantages in detecting DNA sample nucleic acid by using fluorescence PCR.

Table 19.

Table 20.

Table 21.

12.2. Formulation 12-2, formulation 12-3 and formulation 12-4

New sputum samples containing respiratory viruses, gonococci and human cells were tested using the method of section 12.1 according to the following formulations of release reagents with different amounts of components. The result shows that the release reagent components of the application treat samples in different concentration ranges, and the obtained nucleic acid has higher release effect when being detected by fluorescence PCR.

Formula 12-2: 0.01mmol/L of Cymantadine, 0.8% (w/v) of dodecylbenzenesulfonate, 50mmol/L of sodium chloride, 0.05% (v/v) of ethanol and 100mmol/L of sodium hydroxide, and sterilized purified water. The results are shown in table 22 and fig. 15.

TABLE 22 formulation 12-2 test results

Formula 12-3: 0.1mmol/L of Cymantadine, 2% (w/v) of dodecylbenzenesulfonate, 1200 mmol/L of sodium chloride, 0.1% (v/v) of ethanol and 500mmol/L of sodium hydroxide, and sterilized purified water. The results are shown in table 23 and fig. 16.

TABLE 23 test results for formulations 12-3

Formula 12-4: 0.5mmol/L of Cymantadine, 0.01% (w/v) of dodecylbenzenesulfonate, 500mmol/L of sodium chloride, 1% (v/v) of ethanol, and 1250 mmol/L of sodium hydroxide, and sterilized purified water. The results are shown in table 24 and fig. 17.

TABLE 24 formulation 12-4 test results

Example 13 comparative testing of the ability of nucleic acid releasing agents of the invention to treat sputum samples which have been liquefied and added to a preservation solution

The experimental groups of this example were formulated with release agents: the following component concentrations are all prepared with the release agent reagent of the invention at the final concentration after preparation: the following components are prepared with potassium chloride of 100mmol/L, cyperetin of 0.25mmol/L, lithium dodecyl benzene sulfonate of 1% mass-volume ratio, ethanol of 0.5% (v/v), strong base (sodium hydroxide of 500 mmol) and sterilized purified water as solvent.

2 clinical sputum samples (sample No. 1 and sample No. 2) were selected, which contained respiratory viruses, human cells, and gonococci. Liquefaction and storage were carried out by the method of the experimental group in example 7. After the samples were stored for 2 days, the samples were added with the nucleic acid releasing agent and the commercial nucleic acid releasing agent of this example as required for lysis treatment. After the sample is completely processed, six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescence probe method) and gonococcal nucleic acid detection kits (PCR-fluorescence probe method) produced by Shengxiang biological science and technology Co.

The detection results (Ct values) are shown in tables 25 to 27 and FIG. 11, in FIG. 11, A is an RNA amplification curve of respiratory virus No. 1; b is the RNA amplification curve of respiratory virus No. 2; c is a sample No. 1 human cell DNA amplification curve; d is a sample No. 2 human cell DNA amplification curve; e is a No. 1 sample gonococcus DNA amplification curve; f is the sample No. 2 gonococcus DNA amplification curve. As a result, the concentration of nucleic acid detected by six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescent probe method) produced by Santa Clariti Biotechnology Ltd was higher than that of the nucleic acid detected by the commercial release agent after the RNA viral sample with the preservation solution matrix was treated with the nucleic acid release agent in this example. It is demonstrated that the experimental group of this example shows a higher efficiency of treating the RNA virus sample containing the preservation solution matrix with the release agent in the case of nucleic acid detection using fluorescence PCR methodology.

In this example, after treating a DNA sample with a preservation solution matrix with a nucleic acid releasing agent, six respiratory tract pathogen nucleic acid detection kits (PCR-fluorescent probe method) manufactured by Santa Clan Biotechnology Ltd were used to detect that the concentration of DNA nucleic acid was higher than that of a commercial releasing agent. It is demonstrated that the test panel of this example also shows a higher efficiency of treatment of the DNA sample containing the preservative fluid matrix with the release agent in the case of nucleic acid detection using fluorescence PCR methodology.

Table 25.

Table 26.

Table 27.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.

Claims

1. A viscous biological sample liquefaction release combination comprising a liquefaction component and a nucleic acid release component:

the liquefaction component comprises guaifenesin and first strong base, wherein the concentration of the guaifenesin is 20-500 mmol/L, and the concentration of the first strong base is 5-500 mmol/L;

component i) Comprises the following steps: 0.1-2% (v/v) Tween 20, 0.1-3% (v/v) Triton X-100, 0.1-3% (v/v) ethylphenyl polyethylene glycol, and 20-1 mol/L Na⁺And/or K⁺50 mmol/L-1.25 mol/L of second alkali, a first adsorbent and an aqueous solvent;

component ii) comprises: 0.01 mmol/L-0.5 mmol/L surfactant, 0.01% -2% (w/v) dodecylbenzene sulfonate, 50 mmol/L-1.2 mol/L Na⁺and/K⁺0.05% -1% (v/v) ethanol, 100 mmol/L-1.25 mol/L third alkali, and optionally a second adsorbent.

2. The combination of claim 1 wherein the first adsorbent is a Chelex resin at a concentration of 1% to 15% (w/v) in component i); and/or the presence of a catalyst in the reaction mixture,

the second adsorbent is Chelex resin, and the concentration of the Chelex resin in the component ii) is 1-15% (w/v).

3. The combination product of claim 1, wherein the first adsorbent comprises 0.5 to 1mmol/L trehalose.

4. A combination according to claim 1, wherein component ii) comprises: 0.01 mmol/L-0.5 mmol/L surfactant, 0.01% -1% (w/v) dodecylbenzene sulfonate, 60 mmol/L-1 mol/L Na⁺and/K⁺0.05% -1% (v/v) ethanol, 150 mmol/L-1.25 mol/L third strong alkali and a second adsorbent.

5. A combination product according to any of claims 1 to 4, wherein the liquefaction component further comprises rigid microparticles.

6. A combination product according to any of claims 1 to 4, wherein the first strong base in the liquefaction component is at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide and choline; and/or the presence of a gas in the gas,

the second strong base in component i) is at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide and choline; and/or the presence of a gas in the gas,

the third strong base in component ii) is at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, and choline; and/or the presence of a gas in the gas,

the aqueous solvent in the component i) is Tris-HCl with the concentration of 0.5-500 mmol/L; and/or the presence of a gas in the gas,

the surfactant in the component ii) is one or more of sargentin, sodium dodecyl sulfate and ethylenediamine tetraacetic acid.

7. A combination product according to any of claims 1 to 4, further comprising a preservative component;

the preservation component comprises the following components:

a) the buffer component is used for adjusting the pH value of the preservation system to 6-8;

b) an osmotic pressure regulating component; and

c) at least one of trehalose, mannitol, and glycerol.

8. The combination of claim 7, wherein the preservation component comprises: 1 mmol/L-5 mmol/L citric acid, 0.1% -1.2% (w/v) sodium chloride, 0.1% -1.2% (w/v) potassium chloride, 0.8 mol/L-1 mol/L glycine, 0.6 mol/L-1 mol/L isoleucine, 0.5 mol/L-1 mol/L trehalose, 1.5% -4.5% (w/v) mannitol and 2% -10% (v/v) glycerol.

9. A kit comprising the combination product of any one of claims 1 to 8.

10. The kit of claim 9, further comprising one or more of an extraction agent, an amplification agent, and a detection agent for nucleic acids.

11. A method for liquefying and releasing a viscous biological sample, comprising the steps of:

1) mixing a viscous biological sample with the liquefaction component of the viscous biological sample liquefaction release combination product of any of claims 1-8 to obtain a first mixture;

2) incubating the first mixture to obtain a second mixture;

3) mixing the second mixture with the nucleic acid releasing component of the viscous biological sample liquefaction release combination product of any of claims 1-8 and releasing nucleic acid in the sample.

12. The method of claim 11, wherein the pH of the first mixture is 10 or more.

13. The method of claim 11, wherein the concentration of guaifenesin in the first mixture is from 1mmol/L to 1 mol/L.

14. The method of claim 11, wherein in the step 2), the incubation temperature is 18 ℃ to 35 ℃, and/or the incubation time is not less than 5 min.

15. The method of claim 11, wherein the step 2) further comprises: mixing the incubated first mixture with a preservation component for preservation;

the preservation component is as defined in the viscous biological sample liquefaction release combination product of claim 7 or 8.

16. The method of claim 15, wherein the volume ratio of the preservation component to the first mixture is 1: (2-4).

17. The method of claim 11, wherein the volume ratio of the second mixture to the nucleic acid releasing component in step 3) is 1: (0.5-1.5).

18. The method of claim 11, wherein the viscous biological sample is sputum and/or cervical mucus.

19. The method for extracting nucleic acid from the viscous biological sample comprises the following steps:

releasing nucleic acids from the viscous biological sample using the method of any one of claims 11-18;

extracting the released nucleic acids using a nucleic acid extracting agent.

20. A method for amplifying nucleic acids in a viscous biological sample, comprising the following step 1), with or without the following step 2), and comprising the following step 3):

1) releasing nucleic acids from the viscous biological sample using the method of any one of claims 11-18;

2) extracting the released nucleic acids using a nucleic acid extracting agent;

3) amplifying the nucleic acid using a nucleic acid amplification agent.

21. A method for detecting nucleic acid in a viscous biological sample, comprising the following step 1), with or without the following step 2), with or without the following step 3), and comprising the following step 4):

2) extracting the released nucleic acids using a nucleic acid extracting agent;

3) amplifying the nucleic acid using a nucleic acid amplification agent;

4) detecting the nucleic acid using a nucleic acid detection agent;

the detection methods are for non-diagnostic and therapeutic purposes.