Liquefying agent, application thereof and nucleic acid detection system

Abstract

The invention discloses a liquefying agent, application thereof and a nucleic acid detection system, the liquefying agent comprises strong base, acetylcysteine and 2-(N-morpholine) 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 to 300 mmol / L; the concentration of the 2-(N-morpholine) ethanesulfonic acid ranges from 2 mmol / L to 5 mmol / L. Through the synergistic effect of the strong base, the acetylcysteine and the 2-(N-morpholine) ethanesulfonic acid, the liquefaction time can be remarkably shortened, the viscous biological sample treatment efficiency and speed can be improved, and the viscous biological sample treated by the liquefier can be directly used for nucleic acid amplification; and the viscous biological sample treated by the liquefying agent can be stored for a long time, the accuracy and reliability of nucleic acid detection are ensured by long-time stability, and the credibility of a detection result is improved.

Description

Technical Field

[0001] The invention belongs to the field of medical sample processing, and specifically relates to a liquefier and its application and a nucleic acid detection system. Background Art

[0002] The liquefaction technology of viscous biological samples is an important direction in the fields of medicine and biomedicine, and is mainly used for the pretreatment of viscous biological samples. At present, the common methods for processing viscous biological-sputum samples are sodium hydroxide method, DTT method and protease method: 1. Sodium hydroxide method: Use high concentration sodium hydroxide to quickly dilute mucus at room temperature. The existing technology uses high concentration sodium hydroxide method for sample processing. After the sample is liquefied and diluted, the supernatant is discarded, and physiological saline is used to repeatedly blow and wash, enrich and extract. However, the defect of this method is that the biological cell membrane is extremely fragile and breaks quickly when it comes into contact with strong alkali and strong acid. During the treatment process of sodium hydroxide method, cells in the sample are prone to rupture, and nucleic acids are released in the supernatant, resulting in nucleic acid loss and unstable test results. 2. DTT method: Add 4 times the volume of 0.1% DTT (dithiothreitol) solution to the sample, place it for 15 minutes, shake and mix until the mucus is homogenized, which is a more traditional method that uses sulfhydryl cleavage protein. The disadvantages of this method are: it is time-consuming and the cost of DTT is high, the solution state is poorly stable, it becomes ineffective after being oxidized in a week, and it has a pungent odor and is toxic, making it unsuitable for medical testing and processing large quantities of samples. 3. Protease method: The existing technology adds a large volume of buffer and proteinase K to the sample and incubates at 56°C for 4 to 12 hours to ensure the release of nucleic acids in the sample, but the time is too long, which increases labor costs and the large volume leads to serious sample dilution. Summary of the invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a liquefaction agent and its application and nucleic acid detection system, so as to realize the rapid liquefaction of viscous biological samples and directly use them for nucleic acid amplification after liquefaction.

[0004] The invention provides a liquefier, which comprises a strong base, acetylcysteine ​​and 2-(N-morpholine)ethanesulfonic acid. In the liquefier, 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-morpholine)ethanesulfonic acid is 2 mmol / L to 5 mmol / L.

[0005] The liquefier of the present invention, on the one hand, effectively reduces the viscosity of acidic mucin in the viscous biological sample by a strong base, realizes rapid liquefaction, and then the sulfhydryl group of the thiol reducing agent, acetylcysteine, is exchanged with the disulfide bond of the mucin, thereby cleaving the mucin molecule, and at the same time has a certain cleavage effect on the deoxyribonucleic acid fiber, thereby reducing the viscosity of the viscous biological sample. Through the synergistic effect of the strong base and acetylcysteine, the viscosity of the viscous biological sample is effectively reduced, and the liquefaction efficiency of the viscous biological sample is improved. At the same time, the concentration of the strong base used is only 0.2mol / L to 1mol / L, and the concentration of the strong base used is significantly reduced, so that the stability of the nucleic acid released during the liquefaction process of the viscous biological sample and after liquefaction is improved. On the other hand, the liquefaction process of viscous biological samples will also break up 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-morpholine)ethanesulfonic acid, can not only serve as a pH buffer to stabilize the pH value of the system, but also maintain the stability of nucleic acids in the liquefaction solution, so that nucleic acids can be effectively protected from degradation during and after the liquefaction process of the viscous biological samples, thereby significantly improving the detection accuracy. It can also serve as a surfactant to release more nucleic acids into the viscous biological sample liquefaction solution and improve the detection sensitivity. Through the synergistic effect of strong alkali, acetylcysteine ​​and 2-(N-morpholine)ethanesulfonic acid, the liquefaction time can be significantly shortened, the efficiency and speed of viscous biological sample processing can be improved, and the viscous biological samples treated with the liquefier can be directly used for nucleic acid amplification. The viscous biological samples treated with the liquefier can be stored for a long time, and the long-term stability ensures the accuracy and reliability of nucleic acid detection and improves the credibility of the detection results.

[0006] Preferably, in the liquefier, the concentration of the strong base is 0.25 mol / L to 0.75 mol / L.

[0007] In this embodiment, in the liquefier, the concentration of the strong base is 0.25 mol / L to 0.75 mol / L. Within this range, the strong base concentration can effectively reduce the viscosity of acidic mucus proteins in viscous biological samples to achieve rapid liquefaction, and reduce the unstable effects of the strong base on the liquefaction process and the nucleic acids released thereafter.

[0008] Preferably, in the liquefier, the concentration of acetylcysteine ​​is 15 mmol / L to 45 mmol / L.

[0009] In the present solution, the concentration of acetylcysteine ​​in the liquefier is 15 mmol / L to 45 mmol / L. Within this range, the concentration of acetylcysteine ​​can lyse mucin molecules, thereby reducing the viscosity of sputum. At the same time, the concentration of strong alkali used can be reduced, reducing the damage of strong alkali to nucleic acids, thereby improving the stability of nucleic acids released during the liquefaction process of sputum and after liquefaction.

[0010] Preferably, in the liquefier, the concentration of the 2-(N-morpholine)ethanesulfonic acid is 4 mmol / L to 5 mmol / L.

[0011] Preferably, the strong base includes at least one of sodium hydroxide and potassium hydroxide.

[0012] The invention provides an application of the liquefier in liquefying a viscous biological sample.

[0013] Preferably, the viscous biological sample comprises at least one of sputum, cervical mucus, genital swab, and lavage fluid.

[0014] Preferably, the volume ratio of the viscous biological sample to the liquefier is 1:(1-4).

[0015] The present invention also provides a nucleic acid detection system, comprising the liquefier according to any one of claims 1 to 5 and a nucleic acid detection reagent.

[0016] Preferably, the nucleic acid detection reagent comprises at least one of a primer probe, a PCR buffer, a DNA polymerase, an UDG enzyme, and dNTPs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment groups 1A to 4A in Example 1 are compared.

[0018] Figure 2 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1B to 3B in Example 2 are compared.

[0019] Figure 3 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1C to 2C in Example 3 are compared.

[0020] Figure 4 The stability amplification curve comparison diagram of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and comparison group 3A in Comparative Example 1, respectively.

[0021] Figure 5 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and comparison groups 1B to 6B in Comparative Example 2 are compared.

[0022] Figure 6The melting curve comparison diagrams of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment groups 1A to 4A in Example 1 are shown.

[0023] Figure 7 The melting curve comparison diagrams of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1B to 3B in Example 2 are shown.

[0024] Figure 8 The melting curve comparison diagrams of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1C to 2C in Example 3 are shown.

[0025] Fig. 9 The melting curve comparison diagrams of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and comparison group 3A in Comparative Example 1 are shown.

[0026] Fig.10 The melting curve comparison diagrams of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and comparison groups 1B to 6B in Comparative Example 2 are shown.

[0027] Fig.11 The stability amplification curve comparison diagram is a graph of the sputum samples containing Streptococcus pneumoniae treated with the liquefier of treatment group 1A in Example 1 and stored at room temperature for different time periods (0 h, 2 h, 4 h, 6 h, 8 h).

[0028] Fig.12 This is a comparison chart of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefier of treatment group 1A in Example 1 and stored at room temperature for different time periods (0 h, 2 h, 4 h, 6 h, 8 h).

[0029] Fig.13 This is a comparison chart of stability amplification curves of sputum samples containing Streptococcus pneumoniae treated with the liquefier of treatment group 1A in Example 1 and stored at 4° C. for different time periods (0 h, 24 h, 48 h, 72 h).

[0030] Fig.14 This is a comparison of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefier of treatment group 1A in Example 1 and stored at 4 degrees for different time periods (0 h, 24 h, 48 h, 72 h). DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0032] Example 1

[0033] Treatment Group 1A

[0034] 1. The components and their final concentrations in the liquefier of this embodiment are: 500 mmol / L sodium hydroxide, 30 mmol / L acetylcysteine, and 5 mmol / L 2-(N-morpholine)ethanesulfonic acid.

[0035] 2. Method for preparing a liquefier: Sodium hydroxide, acetylcysteine ​​and 2-(N-morpholine)ethanesulfonic acid are weighed separately on a molecular balance, purified water (sterilized) is added to dissolve and adjust the volume to obtain a liquefier.

[0036] Treatment Groups 2A to 4A

[0037] Treatment groups 2A to 4A prepared liquefiers with reference to the liquefier components, final concentrations and preparation methods provided by treatment group 1A, with the concentration of sodium hydroxide used in the liquefier as a variable. The variables used to prepare the liquefiers in treatment groups 2A to 4A are shown in Table 1. Except for the above differences, the operating steps for preparing the liquefiers in treatment groups 2A to 4A were strictly consistent with those in treatment group 1A.

[0038] Table 1 Variables for preparing liquefier in Example 1

[0039] Group Final concentration of sodium hydroxide in the liquefier (mmol / L) Treatment Group 1A 500 Treatment Group 2A 250 Treatment Group 3A 1000 Treatment Group 4A 750

[0040] Example 2

[0041] In this example, the liquefier components and their final concentrations and preparation methods provided by treatment group 1A of Example 1 were used to prepare the liquefier, and the concentration of acetylcysteine ​​used in the liquefier was used as a variable. The variables used to prepare the liquefier in this example are shown in Table 2. Except for the above differences, the operating steps for preparing the liquefier in this example are strictly consistent with those of treatment group 1A.

[0042] Table 2 Variables for preparing liquefier in Example 2

[0043]

[0044]

[0045] Example 3

[0046] In this example, the liquefier components and their final concentrations and preparation methods provided by treatment group 1A of Example 1 were used to prepare the liquefier, and the concentration of 2-(N-morpholine)ethanesulfonic acid used in the liquefier was used as a variable. The variables used to prepare the liquefier in this example are shown in Table 3. Except for the above differences, the operating steps for preparing the liquefier in this example are strictly consistent with those of treatment group 1A.

[0047] Table 3 Variables for preparing liquefier in Example 3

[0048] Group Final concentration of 2-(N-morpholino)ethanesulfonic acid in the liquefier (mmol / L) Treatment Group 1A 5 Treatment Group 1C 2 Treatment Group 2C 4

[0049] Comparative Example 1

[0050] In this comparative example, the liquefier components and their final concentrations and preparation methods provided by treatment group 1A of Example 1 were used to prepare the liquefier, with the components in the liquefier reduced as variables, and the concentrations of the unchanged components were kept the same as those of treatment group 1A. The variables used to prepare the liquefier in this comparative example are shown in Table 4. Except for the above differences, the operating steps for preparing the liquefier in this comparative example were kept the same as those of treatment group 1A.

[0051] Table 4 Variables for preparing liquefier in Comparative Example 1

[0052] Group Components Treatment Group 1A Sodium hydroxide, acetylcysteine, and 2-(N-morpholino)ethanesulfonic acid Comparative Group 1A Acetylcysteine ​​and 2-(N-morpholino)ethanesulfonic acid Comparative Group 2A Sodium hydroxide and 2-(N-morpholino)ethanesulfonic acid Comparative group 3A Sodium hydroxide and acetylcysteine

[0053] Comparative Example 2

[0054] In this comparative example, the liquefier components and their final concentrations and preparation methods provided by treatment group 1A of Example 1 were used to prepare the liquefier, with the replacement of the components in the liquefier and the concentrations of the replaced components as variables, and the unchanged component concentrations were kept the same as those of treatment group 1A. The variables used to prepare the liquefier in this comparative example are shown in Table 5. Except for the above differences, the operating steps for preparing the liquefier in this comparative example were strictly kept the same as those of treatment group 1A.

[0055] Table 5 Variables for preparing liquefier in Comparative Example 2

[0056]

[0057] Performance Testing

[0058] Test Example 1

[0059] Liquefaction effect of liquefiers on sputum samples

[0060] Viscous biological samples: Two clinical sputum samples were selected and divided into 19 portions in parallel, which were recorded as samples 1 to 2. The samples were grade III sputum samples.

[0061] The consistency of sputum samples is registered based on the following classification:

[0062] Grade I sputum is relatively rice soup-like or foamy, and there is no residue when it touches the glass tube after direct suction;

[0063] Grade II sputum is slightly more viscous than Grade I. After suctioning, a small amount of sputum adheres to the glass tube, but it can be easily washed away with water.

[0064] The viscosity of grade III sputum is more viscous than that of grade II, and it may be yellow or green, a darker color. After suction with the suction tube, a large amount of sputum will remain on the wall of the glass tube, which is not easy to be flushed clean by water.

[0065] Experimental method: Add liquefier twice the volume of sputum to the sputum sample, mix thoroughly, place at room temperature, and observe the effect of the liquefied liquid at 5 minutes and 10 minutes.

[0066] Analysis of experimental results: The liquefaction effects of the liquefiers were compared, and the comparison indicators included the complete liquefaction time, whether there was visible sputum or more mucus 10 minutes after the liquefaction time, and whether pipetting operations could be performed (a 10μL pipette was used in this test case).

[0067] Experimental results: The liquefaction effects of 19 liquefiers are shown in Table 6. The results show that in Examples 1 to 3, each liquefier component and its concentration are within the scope of the technical solution to be protected by the present invention, which can achieve the best liquefaction efficiency for sputum samples. And corresponding to the main functions of the strong base and the reducing agent, the higher the concentration of the two, the better the liquefaction effect. In Comparative Example 1, when the components of the strong base and the reducing agent in the liquefier are changed, the effect on the liquefaction effect is great. This result shows that the synergistic effect of the strong base and the thiol reducing agent can improve the liquefaction efficiency of the sputum sample. In Comparative Example 2, when acetylcysteine ​​is replaced by guanidine hydrochloride, the liquefaction effect will be worse. This result shows that different protein denaturants have different effects on the liquefaction of sputum. When the thiol reducing agent is replaced by another reducing agent containing one or two thiol functional groups, the liquefaction effect of the comparison group 2B and the comparison group 3B is equivalent to the liquefier of the treatment group 1A. At the same time, when the types of other surfactants or buffers are changed, the liquefaction effect is equivalent to the liquefier of the treatment group 1A. The same type of reducing agent has little effect on the liquefaction effect of sputum. In addition, since the buffer has little effect on the liquefaction effect of sputum, replacing different surfactants or buffer types will not have much effect on the liquefaction effect of sputum.

[0068] Table 6

[0069]

[0070] In Table 6, basic liquefaction is specifically: basic liquefaction, 10μL pipette can be used to draw out the string; some bubbles appear specifically: some bubbles appear, 10μL pipette can be used to draw out the liquid; and complete liquefaction is specifically: complete liquefaction, 10μL pipette can be used to draw out the liquid. In Table 6, the liquefaction degree is ranked from high to low, in the following order: complete liquefaction > some bubbles appear > basic liquefaction > no liquefaction.

[0071] Test Example 2

[0072] Experimental effect of nucleic acid amplification directly after liquefaction treatment

[0073] Viscous biological samples: Two clinical sputum samples were selected, containing Streptococcus pneumoniae and Staphylococcus aureus respectively. They were divided into two parallel parts, recorded as samples 1 and 2 respectively. The samples were grade II sputum samples.

[0074] Experimental method: Two clinical samples divided into two parts in parallel were liquefied using the liquefiers of the above embodiments and comparative examples respectively. The liquefaction condition is to mix the liquefied liquid with the sample at 2 times the sample volume and let it stand at room temperature for 10 minutes. The six respiratory pathogen nucleic acid detection kits (multiple fluorescence PCR method) (National Medical Device Registration No. 20223400597) of Shengxiang Biotechnology Co., Ltd. were used to perform fluorescence quantitative PCR and melting curve method amplification verification on Hongshi's PCR instrument for the treated samples. Among them, Streptococcus pneumoniae is a fluorescence amplification curve, and Staphylococcus aureus is a melting curve.

[0075] The experimental results are as follows Figures 1 to 10 And as shown in Table 7.

[0076] Figure 1 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment groups 1A to 4A in Example 1 are compared. Figure 1 It can be seen that by changing the concentration of sodium hydroxide in the liquefier, sputum samples can be directly amplified in different treatment groups. However, as the concentration of sodium hydroxide increases to a certain level, the inhibition of the direct amplification system is significantly increased, which is manifested as a decrease in the fluorescence intensity in the plateau phase of the amplification curve.

[0077] Figure 2 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1B to 3B in Example 2 are compared. Figure 2 It can be seen that by changing the concentration of acetylcysteine ​​in the liquefier, sputum samples can be directly amplified in different treatment groups. However, when acetylcysteine ​​increases to a certain concentration, the inhibition of the direct amplification system is significantly increased, which is manifested as a decrease in the fluorescence intensity in the plateau phase of the amplification curve.

[0078] Figure 3 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1C to 2C in Example 3 are compared. Figure 3It can be seen that changing the concentration of 2-(N-morpholino)ethanesulfonic acid in the liquefier has a greater impact on the direct amplification of the liquefier, and affects the Ct value of the sample detection and the fluorescence intensity in the plateau phase. This is because as the 2-(N-morpholino)ethanesulfonic acid gradually increases, it has a certain stabilizing effect on the nucleic acid released in the liquefier.

[0079] Figure 4 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and comparison group 3A in comparative example 1 are compared. Figure 4 It can be seen that after omitting 2-(N-morpholine)ethanesulfonic acid from the liquefier component, the Ct value of the nucleic acid detection amplification curve is delayed to a certain extent, and the curve is low. Therefore, in the combination of strong base and acetylcysteine, a certain nucleic acid stabilizer or pH buffer needs to be added to maintain the stability of the nucleic acid in the solution and ensure the detection ability of the nucleic acid after liquefaction.

[0080] Figure 5 The stability amplification curves of direct amplification detection of sputum samples containing Streptococcus pneumoniae treated with the liquefiers of treatment group 1A in Example 1 and comparison groups 1B to 6B in Comparative Example 2 are compared. Figure 5 It can be seen that replacing acetylcysteine ​​with other protein denaturants or thiol reducing agents, and using different surfactants or pH buffers will have a significant impact on the direct amplification detection of liquefiers. This may be due to the strong inhibition of these reagents on the nucleic acid amplification system.

[0081] Figure 6 The melting curve comparison diagram of the direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment groups 1A to 4A in Example 1 is shown. Figure 6 It can be seen that by changing the concentration of sodium hydroxide in the liquefier, sputum samples can be directly amplified in different treatment groups. However, as the concentration of sodium hydroxide increases to a certain level, the inhibition of the direct amplification system increases significantly, which is manifested as a decrease in the peak value of the melting curve.

[0082] Figure 7 The melting curve comparison diagram of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1B to 3B in Example 2 is shown. Figure 7 It can be seen that by changing the concentration of acetylcysteine ​​in the liquefier, sputum samples can be directly amplified in different treatment groups. However, when acetylcysteine ​​rises to a certain concentration, the inhibition of the direct amplification system is significantly increased, which is manifested as a decrease in the peak value of the melting curve.

[0083] Figure 8The melting curve comparison diagram of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and treatment groups 1C to 2C in Example 3 is shown. Figure 8 It is clear that changing the concentration of 2-(N-morpholine)ethanesulfonic acid in the liquefier also has a significant effect on the peak of the melting curve.

[0084] Fig. 9 The melting curve comparison diagram of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and comparison group 3A in Comparative Example 1 is shown. Fig. 9 It can be seen that when 2-(N-morpholine)ethanesulfonic acid is omitted from the liquefier, the characteristic peak of the target specific product does not appear in the melting curve, and the liquefied product cannot be directly used for nucleic acid amplification.

[0085] Fig.10 The melting curve comparison diagram of direct amplification detection of sputum samples containing Staphylococcus aureus treated with the liquefiers of treatment group 1A in Example 1 and comparison groups 1B to 6B in Comparative Example 2 is shown. Fig.10 It can be seen that replacing acetylcysteine ​​with other protein denaturants or thiol reducing agents, using different surfactants or pH buffers will also have a significant impact on the direct expansion melting curve detection of the liquefaction agent.

[0086] Table 7

[0087]

[0088]

[0089] In Table 7, the larger the Rm, the better the direct amplification effect; “ / ” means that no signal value was detected or no melting curve peak was found, and the result was negative.

[0090] Test Example 3

[0091] Effect of preservation of pathogens at room temperature after processing sputum samples

[0092] Viscous biological samples: A clinical sputum sample containing Streptococcus pneumoniae and Staphylococcus aureus was selected. The sample was a grade II sputum sample. The sample was divided into 5 equal parts.

[0093] Experimental method: The clinical samples were divided into 5 parts in parallel and liquefied with a liquefier. The liquefaction condition was to mix the liquefier with the sample at 2 times the sample volume and let it stand at room temperature for 0h, 2h, 4h, 6h, and 8h respectively. The six respiratory pathogen nucleic acid detection kits (multiple fluorescence PCR method) (National Medical Device Registration No. 20223400597) of Shengxiang Biotechnology Co., Ltd. were used to perform fluorescence quantitative PCR and melting curve method amplification verification on the processed samples.

[0094] The experimental results are as follows Fig.11 , Fig.12 As shown in Table 8, after the selected sputum samples were liquefied and placed at room temperature for different periods of time, when the samples treated with the liquefier of the present invention were subjected to extraction-free amplification using the fluorescent quantitative PCR method and the melting curve method, the preservation of the samples after being placed at room temperature for 8 hours after liquefaction was still no significantly different from that just after liquefaction, indicating that the liquefier provided by the present invention has little effect on the integrity of the pathogen and the pathogen nucleic acid.

[0095] Fig.11 The stability amplification curve comparison diagram is a graph of the sputum samples containing Streptococcus pneumoniae treated with the liquefier of treatment group 1A in Example 1 and stored at room temperature for different time periods (0 h, 2 h, 4 h, 6 h, 8 h).

[0096] Fig.12 This is a comparison chart of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefier of treatment group 1A in Example 1 and stored at room temperature for different time periods (0 h, 2 h, 4 h, 6 h, 8 h).

[0097] Table 8 Test of storage time of liquefaction treatment of treatment group 1A at room temperature

[0098]

[0099] Effect of liquefaction agents on the preservation of pathogens after processing sputum samples at 4°C

[0100] Viscous biological samples: A clinical sputum sample containing Streptococcus pneumoniae and Staphylococcus aureus was selected. The sample was a grade II sputum sample. The sample was divided into 4 equal parts.

[0101] Experimental method: The clinical samples divided into 4 parts were liquefied with a liquefier. The liquefaction condition was to mix the sample with liquefier in a volume of 2 times the sample volume and let it stand at room temperature for 0h, 24h, 48h, and 72h. The six respiratory pathogen nucleic acid detection kits (multiple fluorescence PCR method) (National Medical Device Registration No. 20223400597) of Shengxiang Biotechnology Co., Ltd. were used to perform fluorescence quantitative PCR and melting curve method amplification verification on the processed samples.

[0102] The experimental results are as follows Fig.13 , Fig.14As shown in Table 9, after the selected sputum samples were liquefied and placed at 4°C for different periods of time, when the samples treated with the liquefier of the present invention were subjected to extraction-free amplification using the fluorescent quantitative PCR method and the melting curve method, the preservation of the samples after liquefaction and placing at 4°C for 48 hours was still no different from that just after liquefaction, and the Ct was delayed to a certain extent and the peak height decreased to a certain extent after placing at 4°C for 72 hours. This indicates that the liquefier provided by the present invention has little effect on the integrity of pathogens and pathogen nucleic acids within 48 hours at 4°C.

[0103] Fig.13 This is a comparison chart of stability amplification curves of sputum samples containing Streptococcus pneumoniae treated with the liquefier of treatment group 1A in Example 1 and stored at 4° C. for different time periods (0 h, 24 h, 48 h, 72 h).

[0104] Fig.14 This is a comparison of melting curves of sputum samples containing Staphylococcus aureus treated with the liquefier of treatment group 1A in Example 1 and stored at 4° C. for different time periods (0 h, 24 h, 48 h, 72 h).

[0105] Table 9 Test of storage time of liquefaction treatment of treatment group 1A at 4°C

[0106] 0h 24h 48h 72h Streptococcus pneumoniae-Ct 30.32 30.32 30.25 38.32 Staphylococcus aureus-Tm 69.84 69.91 69.85 69.83

[0107] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A liquefier, characterized in that: The liquefier comprises a strong base, acetylcysteine ​​and 2-(N-morpholine)ethanesulfonic acid); in the liquefier, 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; the concentration of the 2-(N-morpholine)ethanesulfonic acid is 2 mmol / L to 5 mmol / L.

2. The liquefier according to claim 1, characterized in that: In the liquefier, the concentration of the strong base is 0.25 mol / L to 0.75 mol / L.

3. The liquefier according to claim 1, characterized in that: In the liquefier, the concentration of acetylcysteine ​​is 15 mmol / L to 45 mmol / L.

4. The liquefier according to claim 1, characterized in that: In the liquefier, the concentration of the 2-(N-morpholine)ethanesulfonic acid is 4 mmol / L to 5 mmol / L.

5. The liquefier according to claim 1, characterized in that: The strong base includes at least one of sodium hydroxide and potassium hydroxide.

6. A use of a liquefier in liquefying a viscous biological sample, characterized in that: The liquefier comprises the liquefier according to any one of claims 1 to 5.

7. The use of the liquefier according to claim 6 in liquefying a viscous biological sample, characterized in that: The viscous biological sample includes at least one of sputum, cervical mucus, genital swab, and lavage fluid.

8. The use of the liquefier according to claim 7 in liquefying a viscous biological sample, characterized in that: The volume ratio of the viscous biological sample to the liquefier is 1:(1-4).

9. A nucleic acid detection system, characterized in that: The method comprises a liquefying agent as claimed in any one of claims 1 to 5 and a nucleic acid detection reagent.

10. The nucleic acid detection system according to claim 9, characterized in that: The nucleic acid detection reagent includes at least one of a primer probe, a PCR buffer, a DNA polymerase, an UDG enzyme, and dNTPs.

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