Liquefying agent and use thereof, and nucleic acid detection system

Abstract

Provided are a liquefying agent and the use thereof, and a nucleic acid detection system. The liquefying agent comprises a strong base, acetylcysteine, and 2-(N-morpholino)ethanesulfonic acid. In the liquefying agent, the concentration of the strong base is 0.2-1 mol / L; the concentration of the acetylcysteine is 3-300 mmol / L; and the concentration of the 2-(N-morpholino)ethanesulfonic acid is 2-5 mmol / L. By means of the synergistic effect of the strong base, acetylcysteine and 2-(N-morpholino)ethanesulfonic acid, the liquefaction time can be significantly shortened, thereby improving the efficiency and speed of treating viscous biological samples. Furthermore, the viscous biological samples treated with the liquefying agent can be directly used for nucleic acid amplification and have a long storage period. Long-term stability ensures the accuracy and reliability of nucleic acid detection, thereby enhancing the credibility of detection results.

Description

A liquefying agent and its application in nucleic acid detection system Technical Field

[0001] This disclosure pertains to the field of medical sample processing, specifically relating to a liquefying agent and its application in a nucleic acid detection system. Background Technology

[0002] Liquefaction technology for viscous biological samples is an important area in medicine and biomedicine, primarily used for the pretreatment of viscous biological samples. Common methods for processing viscous biological samples—sputum—currently include the sodium hydroxide method, the DTT method, and the protease method: I. Sodium Hydroxide Method: This method uses high-concentration sodium hydroxide to rapidly dilute the mucus at room temperature. Current techniques use this method for sample processing. After liquefaction and dilution, the supernatant is discarded, and the sample is repeatedly washed with physiological saline for enrichment and extraction. However, this method has drawbacks: biological cell membranes are extremely fragile and rupture rapidly upon contact with strong alkalis and acids. During the sodium hydroxide method, cell rupture is common, releasing nucleic acids into the supernatant, leading to nucleic acid loss and unstable test results. II. DTT Method: This method involves adding 4 volumes of 0.1% DTT (dithiothreitol) solution to the sample, incubating for 15 minutes with shaking to mix until the mucus is homogenized. This is a relatively traditional method that utilizes thiol-based protein cleavage. The drawbacks of this method are: it is time-consuming and DTT is expensive; the solution has poor stability and becomes ineffective after one week due to oxidation; it also has an irritating odor and is toxic, making it unsuitable for processing large batches of samples in medical testing. III. Protease Method: Current technology involves adding a large volume of buffer and proteinase K to the sample and incubating at 56°C for 4–12 hours to ensure the release of nucleic acids from the sample. However, the long incubation time increases labor costs, and the large volume leads to severe sample dilution. Summary of the Invention

[0003] To address the aforementioned technical problems, the purpose of this disclosure is to provide a liquefying agent and its application in a nucleic acid detection system, enabling rapid liquefaction of viscous biological samples and direct use of the liquefied sample for nucleic acid amplification.

[0004] This disclosure provides a liquefying agent comprising a strong base, acetylcysteine, and 2-(N-morpholino)ethanesulfonic acid; wherein the concentration of the strong base in the liquefying agent is 0.2 mol / L to 1 mol / L; the concentration of the acetylcysteine ​​is 3 mmol / L to 300 mmol / L; and the concentration of the 2-(N-morpholino)ethanesulfonic acid is 2 mmol / L to 5 mmol / L.

[0005] The liquefying agent disclosed herein effectively reduces the viscosity of acidic mucoproteins in viscous biological samples through a strong base, achieving rapid liquefaction. Then, through the exchange of disulfide bonds between the thiol groups of acetylcysteine, a thiol reducing agent, and the mucoprotein molecules, the mucoprotein molecules are cleaved. Simultaneously, it has a certain cleavage effect on deoxyribonucleic acid fibers, thereby reducing the viscosity of viscous biological samples. Through the synergistic effect of the strong base and acetylcysteine, the viscosity of viscous biological samples is effectively reduced, improving the liquefaction efficiency. Furthermore, the concentration of the strong base used is only 0.2 mol / L to 1 mol / L, a significantly reduced concentration, which improves the stability of the liquefaction process and the nucleic acids released after liquefaction. On the other hand, the liquefaction process of viscous biological samples can also break down pathogens, causing nucleic acids to be released into the viscous biological sample liquefaction solution. The nucleic acid stabilizer in the viscous biological sample liquefaction reagent, 2-(N-morpholino)ethanesulfonic acid, can not only act as a pH buffer to stabilize the pH value of the system, but also maintain the stability of nucleic acids in the liquefaction solution. This ensures that nucleic acids are effectively protected from degradation during and after the viscous biological sample liquefaction process, thereby significantly improving the detection accuracy. It can also act as a surfactant to release more nucleic acids into the viscous biological sample liquefaction solution, thereby improving the detection sensitivity.

[0006] This disclosure provides an application of the liquefying agent in the liquefaction of viscous biological samples.

[0007] This disclosure also provides a nucleic acid detection system, including the liquefying agent and nucleic acid detection reagent.

[0008] The liquefaction agent provided in this disclosure, through the synergistic effect of a strong base, acetylcysteine, and 2-(N-morpholino)ethanesulfonic acid, can significantly shorten the liquefaction time, improve the efficiency and speed of processing viscous biological samples, and allow viscous biological samples treated with the liquefaction agent to be directly used for nucleic acid amplification. Furthermore, the viscous biological samples treated with the liquefaction agent have a long shelf life, and their long-term stability ensures the accuracy and reliability of nucleic acid detection, thereby improving the credibility of the detection results. Attached Figure Description

[0009] Figure 1 is a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefying agents of treatment groups 1A to 4A in Example 1, respectively.

[0010] Figure 2 is a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefying agents of treatment group 1A in Example 1 and treatment groups 1B to 3B in Example 2, respectively.

[0011] Figure 3 is a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefying agents of treatment group 1A in Example 1 and treatment groups 1C-2C in Example 3, respectively.

[0012] Figure 4 is a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefying agent of treatment group 1A in Example 1 and control group 3A in Comparative Example 1, respectively.

[0013] Figure 5 is a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefying agent of treatment group 1A in Example 1 and control groups 1B to 6B in Comparative Examples 2, respectively.

[0014] Figure 6 is a comparison of melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment groups 1A to 4A in Example 1, respectively.

[0015] Figure 7 is a comparison of melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment group 1A in Example 1 and treatment groups 1B to 3B in Example 2, respectively.

[0016] Figure 8 is a comparison of melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment group 1A in Example 1 and treatment groups 1C-2C in Example 3, respectively.

[0017] Figure 9 is a comparison of melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agent of treatment group 1A in Example 1 and control group 3A in Comparative Example 1, respectively.

[0018] Figure 10 is a comparison of melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agent of treatment group 1A in Example 1 and control groups 1B to 6B in Comparative Examples 2, respectively.

[0019] Figure 11 is a comparison of the stability amplification curves of sputum samples containing Streptococcus pneumoniae treated with the liquefying agent of treatment group 1A in Example 1 and stored at room temperature for different times (0h, 2h, 4h, 6h, 8h).

[0020] Figure 12 is a comparison of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefying agent of treatment group 1A in Example 1 and stored at room temperature for different times (0h, 2h, 4h, 6h, 8h).

[0021] Figure 13 is a comparison of the stability amplification curves of sputum samples containing Streptococcus pneumoniae treated with the liquefying agent of treatment group 1A in Example 1 and stored at 4°C for different times (0h, 24h, 48h, 72h).

[0022] Figure 14 is a comparison of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefying agent of treatment group 1A in Example 1 and stored at 4 degrees for different times (0h, 24h, 48h, 72h). Detailed Implementation

[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0024] The term "strong base" refers to a strongly alkaline compound in a liquefying agent system that can effectively reduce the viscosity of acidic mucus proteins in viscous biological samples and achieve rapid liquefaction of the samples.

[0025] The term "viscous biological sample" refers to a biomedical sample with a certain viscosity that requires liquefaction before it can be better tested for nucleic acid.

[0026] The term "sputum" refers to secretions from the respiratory tract (bronchus, trachea, larynx, nose) or exudate from the alveoli.

[0027] The term "cervical mucus" refers to a sample of viscous secretions from the cervix.

[0028] The term "genital swab" refers to a swab sample collected from the mucous membrane of the genital area, which usually has a certain amount of sticky secretions attached to it.

[0029] The term "irrigation fluid" refers to the liquid sample collected after irrigating specific parts of the human body (such as the respiratory tract, reproductive tract, etc.).

[0030] The term "nucleic acid detection reagent" refers to reagents used in conjunction with liquefying agents to perform nucleic acid detection on viscous biological samples.

[0031] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0032] A liquefying agent is disclosed, comprising a strong base, acetylcysteine, and 2-(N-morpholino)ethanesulfonic acid; wherein the concentration of the strong base in the liquefying agent is 0.2 mol / L to 1 mol / L; the concentration of the acetylcysteine ​​is 3 mmol / L to 300 mmol / L; and the concentration of the 2-(N-morpholino)ethanesulfonic acid is 2 mmol / L to 5 mmol / L.

[0033] In some embodiments, the concentration of the strong base in the liquefying agent can be any value within a range of 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L, 0.55 mol / L, 0.6 mol / L, 0.65 mol / L, 0.7 mol / L, 0.75 mol / L, 0.8 mol / L, 0.85 mol / L, 0.9 mol / L, 0.95 mol / L, 1 mol / L, or any value within a range of two values.

[0034] In some embodiments, the concentration of acetylcysteine ​​in the liquefying agent can be any value within a range of 3 mmol / L, 10 mmol / L, 15 mmol / L, 30 mmol / L, 45 mmol / L, 60 mmol / L, 80 mmol / L, 100 mmol / L, 120 mmol / L, 150 mmol / L, 180 mmol / L, 200 mmol / L, 220 mmol / L, 250 mmol / L, 280 mmol / L, 300 mmol / L, or any value within a range of two such values.

[0035] In some embodiments, the concentration of 2-(N-morpholino)ethanesulfonic acid in the liquefying agent can be any value within a range of 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L, 5 mmol / L, or any value within a range of two such values.

[0036] In some embodiments, the concentration of the strong alkali in the liquefying agent is 0.25 mol / L to 0.75 mol / L.

[0037] In this embodiment, the concentration of the strong alkali in the liquefying agent is 0.25 mol / L to 0.75 mol / L. Within this range, the concentration of the strong alkali can effectively reduce the viscosity of acidic mucoproteins in viscous biological samples, achieving rapid liquefaction, while also reducing the unstable impact of the strong alkali on the liquefaction process and the nucleic acids released afterwards.

[0038] In some embodiments, the concentration of acetylcysteine ​​in the liquefying agent is 15 mmol / L to 45 mmol / L.

[0039] In this embodiment, the concentration of acetylcysteine ​​is 15 mmol / L to 45 mmol / L. Within this concentration range, acetylcysteine ​​can break down mucin molecules, thereby reducing sputum viscosity. Simultaneously, it reduces the concentration of strong alkali used, minimizing its damage to nucleic acids and improving the stability of the sputum liquefaction process and the nucleic acids released after liquefaction.

[0040] In some embodiments, the concentration of 2-(N-morpholino)ethanesulfonic acid in the liquefying agent is 4 mmol / L to 5 mmol / L.

[0041] In some embodiments, the strong base includes at least one of sodium hydroxide and potassium hydroxide.

[0042] This disclosure provides the application of the liquefying agent in the liquefaction of viscous biological samples.

[0043] In some embodiments, the viscous biological sample includes at least one of sputum, cervical mucus, genital swabs, and douche fluid.

[0044] In some embodiments, the volume ratio of the viscous biological sample to the liquefying agent is 1:(1 to 4). Specifically, the volume ratio of the viscous biological sample to the liquefying agent can be any value within a range of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, or any value within a range of pairs of values.

[0045] This disclosure also provides a nucleic acid detection system, including the liquefying agent and nucleic acid detection reagent.

[0046] In some embodiments, the nucleic acid detection reagent includes at least one of primers, probes, PCR buffer, DNA polymerase, UDG enzyme, and dNTPs.

[0047] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0048] Example 1

[0049] Processing group 1A

[0050] 1. The components and their final concentrations in the liquefying agent in this embodiment are: 500 mmol / L sodium hydroxide, 30 mmol / L acetylcysteine, and 5 mmol / L 2-(N-morpholino)ethanesulfonic acid.

[0051] 2. Method for preparing liquefying agent: Weigh sodium hydroxide, acetylcysteine ​​and 2-(N-morpholino)ethanesulfonic acid separately in a molecular balance, add purified water (sterilized) to dissolve and make up to volume to prepare liquefying agent.

[0052] Treatment groups 2A to 4A

[0053] Treatments 2A to 4A prepared liquefaction agents by referring to the liquefaction agent components, final concentrations, and preparation methods provided in treatment 1A, using the concentration of sodium hydroxide used in the liquefaction agent as a variable. The variables used in treatments 2A to 4A for preparing liquefaction agents are shown in Table 1. Apart from the differences mentioned above, the operation steps for preparing liquefaction agents in treatments 2A to 4A are strictly consistent with those in treatment 1A.

[0054] Table 1. Variables in the preparation of the liquefying agent in Example 1

[0055] Example 2

[0056] This embodiment prepares a liquefying agent by referring to the liquefying agent components, their final concentrations, and preparation methods provided in treatment group 1A of Example 1. The concentration of acetylcysteine ​​used in the liquefying agent is used as a variable. The variables used in this embodiment for preparing the liquefying agent are shown in Table 2. Except for the differences mentioned above, the operational steps for preparing the liquefying agent in this embodiment are strictly consistent with those in treatment group 1A.

[0057] Table 2. Variables in the preparation of the liquefying agent in Example 2.

[0058] Example 3

[0059] This embodiment prepares the liquefying agent according to the liquefying agent components, their final concentrations, and the preparation method provided in treatment group 1A of Example 1. The concentration of 2-(N-morpholino)ethanesulfonic acid used in the liquefying agent is used as a variable. The variables used in this embodiment for preparing the liquefying agent are shown in Table 3. Except for the differences mentioned above, the operation steps for preparing the liquefying agent in this embodiment are strictly consistent with those in treatment group 1A.

[0060] Table 3. Variables in the preparation of the liquefying agent in Example 3.

[0061] Comparative Example 1

[0062] This comparative example uses the liquefying agent components, their final concentrations, and preparation methods provided in treatment group 1A of Example 1 to prepare the liquefying agent. The components in the liquefying agent are reduced as variables, while the concentrations of the unchanged components remain consistent with treatment group 1A. The variables used in this comparative example for preparing the liquefying agent are shown in Table 4. Apart from the differences mentioned above, the operational steps for preparing the liquefying agent in this comparative example are consistent with those in treatment group 1A.

[0063] Table 4. Variables in the preparation of liquefying agent in Comparative Example 1

[0064] Comparative Example 2

[0065] This comparative example uses the liquefying agent components, their final concentrations, and preparation methods provided in treatment group 1A of Example 1 to prepare the liquefying agent. The variables are the components that are replaced in the liquefying agent and the concentrations of the replaced components. The concentrations of the unchanged components remain consistent with those in treatment group 1A. The variables used in this comparative example for preparing the liquefying agent are shown in Table 5. Except for the differences mentioned above, the operational steps for preparing the liquefying agent in this comparative example are strictly consistent with those in treatment group 1A.

[0066] Table 5. Variables in the preparation of liquefying agents in Comparative Example 2

[0067] Performance testing

[0068] Test Example 1

[0069] The liquefaction effect of liquefying agents on sputum samples

[0070] Viscous biological samples: Two clinical sputum samples were selected and divided into 19 parallel portions, designated as samples 1-2. The samples were grade III sputum samples.

[0071] The criteria for classifying the consistency of sputum samples are as follows:

[0072] Grade I sputum is relatively rice-water-like or foamy, and leaves no residue when it comes into contact with the glass tube after direct suctioning.

[0073] Grade II sputum is slightly thicker than Grade I sputum. After suctioning, a small amount of sputum will stick to the glass tube, but it can be easily rinsed off with water.

[0074] Grade III sputum is more viscous than Grade II sputum and may be yellow or green, with a darker color. After suctioning, a large amount of sputum will remain on the glass tube wall, which is not easily washed away with water.

[0075] Experimental method: Add 2 times the volume of sputum liquefaction agent to the sputum sample, mix thoroughly, place at room temperature, and observe the liquefaction effect at 5 min and 10 min time points.

[0076] Analysis of experimental results: The liquefaction effect of the liquefying agents was compared. The indicators included the time for complete liquefaction and whether there was visible sputum or a lot of mucus after 10 minutes of liquefaction, so that pipetting could be performed (a 10μL pipette was used in this test case).

[0077] Experimental Results: The liquefaction effects of 19 liquefying agents are shown in Table 6. The results indicate that, within the scope of the technical solutions to be protected in Examples 1-3, the components and concentrations of each liquefying agent can achieve optimal liquefaction efficiency for sputum samples. Furthermore, corresponding to the main functions of the strong base and reducing agent, the higher the concentration of both, the better the liquefaction effect. In Comparative Example 1, changing the components of the strong base and reducing agent in the liquefying agent significantly affected the liquefaction effect, indicating that the synergistic effect of the strong base and the thiol reducing agent can improve the liquefaction efficiency of sputum samples. In Comparative Example 2, replacing acetylcysteine ​​with guanidine hydrochloride resulted in a worse liquefaction effect, indicating that different protein denaturants have different sputum liquefaction effects. When the thiol reducing agent was replaced with another reducing agent containing one or two thiol functional groups, the liquefaction effects of control groups 2B and 3B were comparable to those of treatment group 1A. That is, the same type of reducing agent has little effect on the sputum liquefaction effect. Meanwhile, when other surfactants or buffers were changed, the liquefaction effect was comparable to that of treatment group 1A. Since buffers have little effect on sputum liquefaction, changing the type of surfactant or buffer also has little impact on sputum liquefaction.

[0078] Table 6

[0079] In Table 6, the basic liquefaction is specifically as follows:

[0080] Basic liquefaction is defined as follows: a 10μL pipette can aspirate the liquid and produce a stringy consistency;

[0081] The presence of a small amount of foam indicates that a 10μL pipette can be used for aspiration.

[0082] Complete liquefaction specifically means: complete liquefaction, and a 10μL pipette can be used for aspiration.

[0083] In Table 6, the liquefaction levels are sorted from highest to lowest as follows: complete liquefaction > some foaming > basic liquefaction > no liquefaction.

[0084] Test Example 2

[0085] Experimental results of direct nucleic acid amplification after liquefaction agent treatment.

[0086] Viscous biological samples: Two clinical sputum samples were selected, containing Streptococcus pneumoniae and Staphylococcus aureus, respectively. They were divided into two parallel aliquots, designated as samples 1 and 2. The samples were grade II sputum samples.

[0087] Experimental Methods: Two clinical samples, divided into two parallel subsets, were liquefied using the liquefaction agents described in the above examples and the comparative example, respectively. Liquefaction conditions included mixing the liquefaction solution with twice the sample volume, and incubating at room temperature for 10 minutes. The processed samples were then amplified using quantitative real-time PCR and melting curve methods on a Hongshi PCR instrument using the Six Respiratory Pathogen Nucleic Acid Detection Kit (Multiplex Fluorescent PCR Method) (National Medical Device Registration Certificate 20223400597) from Sansure Biotech Inc. The fluorescence amplification curve was used for Streptococcus pneumoniae, and the melting curve was used for Staphylococcus aureus.

[0088] The experimental results are shown in Figures 1-10 and Table 7.

[0089] Figure 1 shows a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with liquefaction agents from treatment groups 1A to 4A in Example 1. As shown in Figure 1, changing the concentration of sodium hydroxide in the liquefaction agent allows for direct amplification of sputum samples in different treatment groups. However, as the concentration of sodium hydroxide increases to a certain level, the inhibition of the direct amplification system significantly increases, manifested as a decrease in fluorescence intensity during the plateau phase of the amplification curve.

[0090] Figure 2 shows a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefaction agent of treatment group 1A in Example 1 and treatment groups 1B-3B in Example 2, respectively. As shown in Figure 2, changing the concentration of acetylcysteine ​​in the liquefaction agent allows for direct amplification of sputum samples in different treatment groups. However, as the concentration of acetylcysteine ​​increases to a certain level, the inhibition of the direct amplification system significantly increases, manifested as a decrease in fluorescence intensity during the plateau phase of the amplification curve.

[0091] Figure 3 shows a comparison of the stability and amplification curves for direct amplification of sputum samples containing Streptococcus pneumoniae treated with the liquefaction agents of treatment group 1A in Example 1 and treatment groups 1C-2C in Example 3, respectively. As shown in Figure 3, changing the concentration of 2-(N-morpholino)ethanesulfonic acid in the liquefaction agent significantly affects the direct amplification, influencing both the Ct value and the fluorescence intensity during the plateau phase. This is because the gradual increase in 2-(N-morpholino)ethanesulfonic acid plays a stabilizing role on the nucleic acids released in the liquefaction agent.

[0092] Figure 4 shows a comparison of the stability and amplification curves for direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefaction agent of treatment group 1A in Example 1 and control group 3A in Comparative Example 1, respectively. As shown in Figure 4, omitting 2-(N-morpholino)ethanesulfonic acid in the liquefaction agent component results in a delayed Ct value and a lower curve for nucleic acid detection amplification. Therefore, in the combination of strong base and acetylcysteine, it is necessary to add a certain amount of nucleic acid stabilizer or pH buffer to maintain the stability of the nucleic acid in the solution and ensure the detection capability of nucleic acid after liquefaction.

[0093] Figure 5 shows a comparison of the stability and amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefaction agents of treatment group 1A in Example 1 and control groups 1B-6B in Comparative Examples 2, respectively. As shown in Figure 5, replacing acetylcysteine ​​with other protein denaturing agents or thiol reducing agents, and using different surfactants or pH buffers, significantly affects the direct amplification detection of the liquefaction agent. This may be due to the strong inhibition of nucleic acid amplification by these reagents.

[0094] Figure 6 is a comparison of melting curves for direct amplification of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment groups 1A to 4A in Example 1. As shown in Figure 6, changing the concentration of sodium hydroxide in the liquefying agent allows for direct amplification of sputum samples in different treatment groups. However, as the concentration of sodium hydroxide increases to a certain level, the inhibition of the direct amplification system significantly increases, as evidenced by a decrease in the peak value of the melting curve.

[0095] Figure 7 shows a comparison of melting curves for direct amplification of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment group 1A in Example 1 and treatment groups 1B-3B in Example 2, respectively. As shown in Figure 7, changing the concentration of acetylcysteine ​​in the liquefying agent allows for direct amplification of sputum samples in different treatment groups. However, as the concentration of acetylcysteine ​​increases to a certain level, the inhibition of the direct amplification system significantly increases, as evidenced by a decrease in the peak value of the melting curve.

[0096] Figure 8 shows a comparison of melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment group 1A in Example 1 and treatment groups 1C-2C in Example 3, respectively. As can be seen from Figure 8, changing the concentration of 2-(N-morpholino)ethanesulfonic acid in the liquefying agent has a significant impact on the peak value of the melting curve.

[0097] Figure 9 shows a comparison of melting curves for direct amplification of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment group 1A in Example 1 and control group 3A in Comparative Example 1, respectively. As shown in Figure 9, omitting 2-(N-morpholino)ethanesulfonic acid in the liquefying agent resulted in the absence of characteristic peaks of the target-specific product in the melting curves, meaning the sample could not be directly used for nucleic acid amplification after liquefaction.

[0098] Figure 10 shows a comparison of the melting curves for direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefying agents of treatment group 1A in Example 1 and control groups 1B-6B in Comparative Examples 2, respectively. As can be seen from Figure 10, replacing acetylcysteine ​​with other protein denaturing agents or thiol reducing agents, as well as using different surfactants or pH buffers, can significantly affect the detection of the liquefying agent's direct amplification melting curve.

[0099] Table 7

[0100] In Table 7, the larger the Rm, the better the direct amplification effect; " / " indicates that no signal value was detected or no melting curve peak was found, and the result is negative.

[0101] Test Example 3

[0102] Preservation effect of pathogens at room temperature after processing sputum samples

[0103] Viscous biological samples: One clinical sputum sample containing Streptococcus pneumoniae and Staphylococcus aureus was selected. The sample was a grade II sputum consistency sample. The sample was divided into 5 equal parts.

[0104] Experimental Methods: Clinical samples, divided into 5 parallel portions, were liquefied using a liquefaction agent. Liquefaction conditions included mixing the liquefied solution with twice the sample volume, and incubating at room temperature for 0 h, 2 h, 4 h, 6 h, and 8 h, respectively. The processed samples were then validated using quantitative real-time PCR and melting curve amplification methods using the Six Respiratory Pathogen Nucleic Acid Detection Kit (Multiplex Fluorescent PCR Method) (National Medical Device Registration Certificate 20223400597) from Sansure Biotech Inc.

[0105] The experimental results are shown in Figures 11 and 12 and Table 8. After liquefying the selected sputum samples and placing them at room temperature for different times, the samples treated with the liquefaction agent of this disclosure were amplified using the real-time PCR method and the melting curve method without extraction. After being placed at room temperature for 8 hours after liquefaction, the preservation of the samples was still not significantly different from that immediately after liquefaction. This indicates that the liquefaction agent provided in this disclosure has little impact on the integrity of pathogens and their nucleic acids.

[0106] Figure 11 is a comparison of the stability amplification curves of sputum samples containing Streptococcus pneumoniae treated with the liquefying agent of treatment group 1A in Example 1 and stored at room temperature for different times (0h, 2h, 4h, 6h, 8h). The amplification curves at different times highly overlap.

[0107] Figure 12 is a comparison of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefying agent of treatment group 1A in Example 1 and stored at room temperature for different times (0h, 2h, 4h, 6h, 8h).

[0108] Table 8. Test of storage time of liquefaction agent 1A in group 1A at room temperature.

[0109] Test Example 4

[0110] The effect of liquefaction agents on pathogen preservation at 4°C after processing sputum samples.

[0111] Viscous biological samples: One clinical sputum sample containing Streptococcus pneumoniae and Staphylococcus aureus was selected. The sample was a grade II sputum consistency sample. The sample was divided into four equal parts.

[0112] Experimental Methods: Clinical samples, divided into four parallel portions, were liquefied using a liquefaction agent. Liquefaction conditions included mixing the liquefaction agent with twice the sample volume, and incubating at room temperature for 0 h, 24 h, 48 h, and 72 h, respectively. The processed samples were then amplified using quantitative real-time PCR and melting curve methods using the Six Respiratory Pathogen Nucleic Acid Detection Kit (Multiplex Fluorescent PCR Method) (National Medical Device Registration Certificate 20223400597) from Sansure Biotech Inc.

[0113] The experimental results are shown in Figures 13 and 14 and Table 9. After liquefying the selected sputum samples and placing them at 4℃ for different times, when the samples treated with the liquefaction agent of this disclosure were amplified using the real-time PCR method and the melting curve method without extraction, the preservation of the samples after liquefaction and placement at 4℃ for 48 hours was still not significantly different from that immediately after liquefaction. Only after 72 hours did the Ct value show a certain delay and a slight decrease in peak height. This indicates that the liquefaction agent provided in this disclosure has minimal impact on the integrity of pathogens and their nucleic acids within 48 hours at 4℃.

[0114] Figure 13 is a comparison of the stability amplification curves of sputum samples containing Streptococcus pneumoniae treated with the liquefying agent of treatment group 1A in Example 1 and stored at 4°C for different times (0h, 24h, 48h, 72h). The amplification curves of 0h, 24h, and 48h are highly overlapping.

[0115] Figure 14 is a comparison of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefying agent of treatment group 1A in Example 1 and stored at 4°C for different times (0h, 24h, 48h, 72h).

[0116] Table 9. Test of storage time of liquefaction agent 1A in treatment group at 4℃.

[0117] The above embodiments are only used to illustrate the technical solutions of this disclosure and are not intended to limit the scope of protection of this disclosure. Although this disclosure has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this disclosure without departing from the substance and scope of the technical solutions of this disclosure.

Claims

1. A liquefier, characterized by, The liquefying agent includes a strong base, acetylcysteine, and 2-(N-morpholino)ethanesulfonic acid; in the liquefying agent, the concentration of the strong base is 0.2 mol / L to 1 mol / L; the concentration of the acetylcysteine ​​is 3 mmol / L to 300 mmol / L; and the concentration of the 2-(N-morpholino)ethanesulfonic acid is 2 mmol / L to 5 mmol / L.

2. The liquefying agent according to claim 1, characterized in that, In the liquefying agent, the concentration of the strong alkali is 0.25 mol / L to 0.75 mol / L.

3. The liquefying agent according to claim 1 or 2, characterized in that, In the liquefying agent, the concentration of the strong alkali is 0.45 mol / L to 0.55 mol / L.

4. The liquefying agent according to any one of claims 1-3, characterized in that, In the liquefying agent, the concentration of acetylcysteine ​​is 15 mmol / L to 45 mmol / L.

5. The liquefying agent according to any one of claims 1-4, characterized in that, In the liquefying agent, the concentration of acetylcysteine ​​is 28 mmol / L to 32 mmol / L.

6. The liquefying agent according to any one of claims 1-5, characterized in that, In the liquefying agent, the concentration of 2-(N-morpholino)ethanesulfonic acid is 4 mmol / L to 5 mmol / L.

7. The liquefying agent according to any one of claims 1-6, characterized in that, In the liquefying agent, the concentration of 2-(N-morpholino)ethanesulfonic acid is 4.5 mmol / L to 5 mmol / L.

8. The liquefying agent according to any one of claims 1-7, characterized in that, The strong base includes at least one of sodium hydroxide and potassium hydroxide.

9. The application of a liquefying agent in the liquefaction of viscous biological samples, characterized in that, The liquefying agent includes the liquefying agent as described in any one of claims 1 to 8.

10. The application of the liquefying agent according to claim 9 in the liquefaction of viscous biological samples, characterized in that, The viscous biological samples include at least one of sputum, cervical mucus, genital swabs, and douches.

11. The application of the liquefying agent according to claim 9 in the liquefaction of viscous biological samples, characterized in that, The viscous biological sample was sputum.

12. The application of the liquefying agent according to claim 9 in the liquefaction of viscous biological samples, characterized in that, The volume ratio of the viscous biological sample to the liquefying agent is 1:(1-4).

13. The application of the liquefying agent according to claim 9 in the liquefaction of viscous biological samples, characterized in that, The volume ratio of the viscous biological sample to the liquefying agent is 1:(1.5 to 3.5).

14. A nucleic acid detection system, characterized in that, Includes the liquefying agent and nucleic acid detection reagent as described in any one of claims 1 to 8.

15. The nucleic acid detection system according to claim 14, characterized in that, The nucleic acid detection reagent includes at least one of primers / probes, PCR buffer, DNA polymerase, UDG enzyme, and dNTPs.

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