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, carbonate, sodium laurate and isopropanol. In the liquefying agent, the concentration of the strong base is 0.5 mol / L to 4 mol / L, the concentration of the carbonate is 25 mmol / L to 200 mmol / L, the concentration of the sodium laurate is 20 mmol / L to 90 mmol / L, and the volume fraction of the isopropanol is 10% to 30%. In the liquefying agent, the strong base, the carbonate, the sodium laurate and the isopropanol have a synergistic effect, so that the liquefying efficiency of a viscous biological sample can be improved, the nucleic acid amplification effect of a sputum sample treated by the liquefying agent after magnetic bead method nucleic acid extraction is good, and the accuracy, the sensitivity and the precision of nucleic acid detection can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical sample processing, and in particular relates to a liquefying agent and its application and a nucleic acid detection system. Background Art

[0002] Liquefaction technology for viscous biological samples is an important direction in the fields of medicine and biomedicine. It is mainly used for the pretreatment of viscous biological samples. In the process of processing viscous biological samples, traditional chemical dissolution methods (such as sodium hydroxide method, physiological saline method and DTT method) have the following technical problems: (1) Long liquefaction time: Traditional liquefaction agents take a long time to liquefy high-viscosity viscous biological samples, affecting experimental efficiency. (2) Poor liquefaction effect: For high-viscosity viscous biological samples, the liquefaction effect of traditional liquefaction agents is not ideal and often cannot be completely liquefied, resulting in a large amount of residue after liquefaction, affecting subsequent analysis. (3) Poor processing stability: The liquefaction effect of traditional liquefaction agents in different batches of viscous biological samples is inconsistent, and the repeatability is poor, making it difficult to ensure the reliability of subsequent experiments. (4) Impact on nucleic acid detection: Some liquefaction agents (such as strong alkali) will have a significant impact on the pH value of the sample during the liquefaction process, which may interfere with the subsequent nucleic acid detection results. 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 to improve the liquefaction effect of viscous biological samples and the nucleic acid detection results.

[0004] The present invention provides a liquefier, which comprises a strong base, a carbonate, sodium laurate and isopropyl alcohol. In the liquefier, the concentration of the strong base is 0.5 mol / L to 4 mol / L, the concentration of the carbonate is 25 mmol / L to 200 mmol / L, the concentration of the sodium laurate is 20 mmol / L to 90 mmol / L, and the volume fraction of the isopropyl alcohol is 10% to 30%.

[0005] In the technical solution of the present invention, on the one hand, sodium laurate is combined with a strong base and a carbonate, which can not only effectively destroy the mucin in the viscous biological sample and depolymerize it, thereby reducing the viscosity of the viscous biological sample and achieving rapid liquefaction, but also can adjust and stabilize the pH value of the solution, keeping it at alkaline, which helps to further decompose the components in the viscous biological sample and reduce the loss of nucleic acid; on the other hand, sodium laurate is combined with isopropyl alcohol, which can not only dissolve the lipid components in the viscous biological sample, destroy the cell membrane and other lipid structures, and help liquefy the viscous biological sample, but also reduce microbial contamination in the sample and protect the nucleic acid from degradation. At the same time, it can further reduce the surface tension of the liquid, help disperse and dissolve the components in the viscous biological sample, and promote liquefaction. The synergistic effect of the strong base, carbonate, sodium laurate and isopropyl alcohol can not only improve the liquefaction efficiency of the viscous biological sample, but also make the sputum sample treated with the liquefier have a good nucleic acid amplification effect after magnetic bead nucleic acid extraction, which can significantly improve the accuracy, sensitivity and precision of nucleic acid detection.

[0006] Preferably, the concentration of the strong base is 1 mol / L to 2 mol / L.

[0007] The concentration of the strong base described in this scheme is 1 mol / L to 2 mol / L. Within this range, the strong base concentration can effectively reduce the viscosity of acidic mucin in viscous biological samples to achieve rapid liquefaction, while also reducing the destabilizing effects of the strong base on the liquefaction process and the nucleic acids released thereafter.

[0008] Preferably, the concentration of the carbonate is 50 mmol / L to 100 mmol / L.

[0009] Preferably, the concentration of sodium laurate is 30 mmol / L to 60 mmol / L.

[0010] The concentration of sodium laurate in this solution is 30 mmol / L to 60 mmol / L. Within this range, sodium laurate can not only destroy mucin in viscous biological samples, reduce the viscosity of the viscous biological samples, achieve the purpose of liquefaction, but also protect nucleic acids.

[0011] Preferably, the volume fraction of the isopropyl alcohol is 15% to 25%.

[0012] The present invention provides an application of the liquefaction agent in liquefying viscous biological samples.

[0013] Preferably, the viscous biological sample includes 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 provides a nucleic acid detection system, comprising the liquefier and a nucleic acid detection reagent.

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

[0017] 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 part of the embodiments of the present invention, rather than all the embodiments.

[0018] Example 1

[0019] Treatment group 1A

[0020] 1. The components and their final concentrations in the liquefier of this embodiment are: 2000 mmol / L sodium hydroxide, 45 mmol / L sodium laurate, 100 mmol / L sodium carbonate and 15% volume fraction (v / v) of isopropyl alcohol.

[0021] 2. Method for preparing a liquefier: Sodium hydroxide, sodium laurate, and sodium carbonate are weighed separately on a molecular balance, isopropyl alcohol is added, and finally purified water (sterilized) is added to dissolve and adjust the volume to obtain a liquefier.

[0022] Treatment groups 2A to 4A

[0023] Treatment groups 2A to 4A prepared liquefiers with reference to the liquefier components, final concentrations, and preparation methods provided in treatment group 1A, with the concentrations of sodium hydroxide and sodium carbonate used in the liquefier as variables. The variables used to prepare the liquefiers in treatment groups 2A to 4A are shown in Table 1. Except for the above differences, the operational procedures for preparing the liquefiers in treatment groups 2A to 4A were strictly consistent with those in treatment group 1A.

[0024] Table 1 Variables for preparing liquefier in Example 1

[0025]

[0026] Example 2

[0027] This example prepared a liquefier using the liquefier components, final concentrations, and preparation methods provided in Treatment Group 1A of Example 1, with the sodium laurate concentration used in the liquefier as a variable. The variables used to prepare the liquefier in this example are shown in Table 2. Aside from the aforementioned differences, the steps for preparing the liquefier in this example were identical to those for Treatment Group 1A.

[0028] Table 2 Variables for preparing liquefier in Example 2

[0029] Group Final concentration of sodium laurate in the liquefier (mmol / L) Treatment group 1A 45 Treatment group 1B 20 Treatment group 2B 90 Treatment group 3B 70

[0030] Example 3

[0031] This example prepared a liquefier using the liquefier components, final concentrations, and preparation methods provided in Treatment Group 1A of Example 1. The volume fraction of isopropyl alcohol used in the liquefier was used as a variable. The variables used to prepare the liquefier in this example are shown in Table 3. Aside from the aforementioned differences, the steps for preparing the liquefier in this example were identical to those for Treatment Group 1A.

[0032] Table 3 Variables for preparing liquefier in Example 3

[0033]

[0034]

[0035] Comparative Example 1

[0036] This comparative example prepared a liquefier using the liquefier components, final concentrations, and preparation method provided in Treatment Group 1A of Example 1. The liquefier components were reduced as variables, while the concentrations of the remaining components remained the same as those in Treatment Group 1A. The variables used to prepare the liquefier in this comparative example are shown in Table 4. Aside from the aforementioned differences, the procedures for preparing the liquefier in this example were strictly consistent with those in Treatment Group 1A.

[0037] Table 4 Variables for preparing liquefier in Comparative Example 1

[0038] Group Components Treatment group 1A Sodium hydroxide, sodium carbonate, isopropyl alcohol, and sodium laurate Comparative group 1A Sodium carbonate, isopropyl alcohol, and sodium laurate Comparative group 2A Sodium hydroxide, sodium carbonate, and isopropyl alcohol Comparative group 3A Sodium hydroxide, sodium laurate, and isopropyl alcohol

[0039] Comparative Example 2

[0040] This comparative example prepared a liquefier using the liquefier components, final concentrations, and preparation method provided in Treatment Group 1A of Example 1. Variables were used, including the replacement of components in the liquefier and the concentrations of the replaced components. The concentrations of the unchanged components remained the same as those in Treatment Group 1A. The variables used to prepare the liquefier in this comparative example are shown in Table 5. Aside from the aforementioned differences, the procedures for preparing the liquefier in this comparative example were strictly consistent with those in Treatment Group 1A.

[0041] Table 5 Variables for preparing liquefier in Comparative Example 2

[0042] Group Components and their concentrations before and after replacement Control group 1B 45mmol / L sodium palmitate replaces 45mmol / L sodium laurate Control group 2B 0.5% (w / v, g / mL) Triton X-100 replaces 45mmol / L sodium laurate Control group 3B 0.5% (v / v) sodium dodecyl sulfate (SDS) replaces 45mmol / L sodium laurate Control group 4B 1.47mmol / L potassium dihydrogen phosphate and 9.38mmol / L disodium hydrogen phosphate replace 100mmol / L sodium carbonate Control group 5B 15% volume fraction ethanol replaces 15% volume fraction isopropanol Control group 6B 15% volume fraction of polyethylene glycol replaces 15% volume fraction of isopropyl alcohol

[0043] Comparative Example 3

[0044] 1. The components and their final concentrations in the liquefier of this embodiment are: 4500 mmol / L sodium hydroxide, 45 mmol / L sodium laurate, 225 mmol / L sodium carbonate and 15% isopropyl alcohol by volume (v / v).

[0045] 2. Preparation of liquefier: Same as treatment group 1A in Example 1.

[0046] Comparative Example 4

[0047] This comparative example prepared a liquefier using the liquefier components, final concentrations, and preparation methods provided in Treatment Group 1A of Example 1. The sodium laurate concentration used in the liquefier was used as a variable. The variables used to prepare the liquefier in this comparative example are shown in Table 6. Aside from the aforementioned differences, the steps for preparing the liquefier in this example were identical to those for Treatment Group 1A.

[0048] Table 6 Variables for preparing liquefier in Comparative Example 4

[0049] Group Final concentration of sodium laurate in the liquefier (mmol / L) Treatment group 1A 45 Control group 1C 100 Control group 2C 10

[0050] Comparative Example 5

[0051] The volume fraction of isopropyl alcohol in the liquefier of this comparative example is: 35% (v / v) isopropyl alcohol, and the rest is the same as that of treatment group 1A in Example 1.

[0052] Performance Testing

[0053] Test Example 1

[0054] Liquefaction effect of liquefier on sputum samples

[0055] Viscous biological samples: Two clinical sputum samples were selected and divided into 23 parallel portions, designated as samples 1 and 2. Sample 1 was a grade II sputum sample, and sample 2 was a grade III sputum sample.

[0056] Sputum sample consistency is registered based on the following classification criteria:

[0057] Grade I sputum is relatively rice-water-like or foamy, and there is no residue when it comes into contact with the glass tube after direct suction;

[0058] Grade II sputum is slightly thicker than Grade I. After suctioning, a small amount of sputum adheres to the glass tube, but it can be easily washed away with water.

[0059] The viscosity of grade III sputum is more viscous than that of grade II, and it may be yellow or green, with a darker color. After suction with a suction tube, a large amount of sputum will remain on the wall of the glass tube, which is not easy to be washed away by water.

[0060] Experimental method: Add liquefaction agent twice the volume of sputum to the sputum sample, mix thoroughly, place at room temperature, and observe the effect of liquefaction at time points such as 5 minutes and 15 minutes.

[0061] Analysis of experimental results: The liquefaction effects of 23 liquefiers were compared based on indicators including the time to complete liquefaction, the presence of visible sputum or a large amount of mucus 15 minutes after liquefaction, and the ability to perform pipetting (a 10 μL pipette was used in this test).

[0062] The experimental results are shown in Table 7: The liquefaction effects of 23 liquefiers are shown in Table 7. The results show that, as can be seen from Examples 1 to 3, each component of the liquefier (strong base, carbonate, sodium laurate, and isopropyl alcohol) has a good liquefaction effect on sputum samples within a certain concentration range (strong base concentration of 0.5-4 mol / L, carbonate concentration of 25-200 mmol / L, sodium laurate concentration of 20-90 mmol / L, and isopropyl alcohol volume fraction of 10%-30%). In addition, compared with treatment group 1A of Example 1, comparison groups 1A-2A of comparative example 1 and comparison group 2C of comparative example 4 show that the liquefaction effect deteriorates when the concentration of sodium hydroxide and sodium laurate is lower or even absent. Therefore, this shows that sodium hydroxide and sodium laurate play an important role in liquefying sputum. As can be seen from Comparative Examples 2, comparison group 4B uses potassium dihydrogen phosphate and disodium hydrogen phosphate as buffer solution, and the liquefaction time difference is not significant, indicating that the impact of buffer solution on liquefaction effect is less. Comparison group 5B and comparison group 6B use ethanol and polyethylene glycol as alcohol reagent to replace the isopropyl alcohol in embodiment 1 treatment group 1A respectively, and there is difference with embodiment 1 treatment group 1A liquefier in liquefaction effect, and the liquefaction effect of ethanol and polyethylene glycol is inferior to isopropyl alcohol. Comparison group 1B uses sodium palmitate to replace the sodium laurate in embodiment 1 treatment group 1A, and the liquefaction effect difference is significant, which shows that the composition of different fatty acid sodiums has a certain impact on the liquefaction effect of liquefier. In addition, comparison group 2B and comparison group 3B are obviously weaker than experimental group liquefier on the liquefaction effect of sputum by using different nonionic surfactants and anionic surfactants to replace the liquefier of sodium laurate, which shows that the surfactant of sodium laurate plays a vital role in this liquefier component.

[0063] Table 7

[0064]

[0065]

[0066] In Table 7, basic liquefaction is defined as: basic liquefaction, stringing can be observed with a 10μL pipette; slight foaming is defined as: slight foaming, a 10μL pipette can be used; and complete liquefaction is defined as: complete liquefaction, a 10μL pipette can be used. In Table 7, the order of liquefaction degree from highest to lowest is: complete liquefaction > slight foaming > basic liquefaction > visible sputum > no liquefaction.

[0067] Test Example 2

[0068] Verification of nucleic acid detection experimental results of liquefied agents

[0069] Viscous biological specimens: A clinical sputum specimen containing Streptococcus pneumoniae was collected and divided into 17 parallel aliquots. The specimen had a grade II consistency.

[0070] Experimental method: A clinical sample divided into 17 parts in parallel was liquefied with a liquefaction agent. The liquefaction condition is to mix the liquefaction liquid with the sample at 2 times the sample volume and let it stand at room temperature for 5 minutes. The nucleic acid extraction or purification reagent S10015 (Xiangchang Medical Equipment No. 20150021) of Shengxiang Biotechnology Co., Ltd. was used to extract nucleic acid from pathogens in sputum samples in a semi-automatic nucleic acid extraction (see the instructions for specific operation steps). The extracted nucleic acid was amplified and verified by fluorescent quantitative PCR using the six respiratory pathogen nucleic acid detection kit (multiple fluorescence PCR method) (National Medical Equipment No. 20223400597) of Shengxiang Biotechnology Co., Ltd. (see the instructions for specific operation steps).

[0071] The experimental results are shown in Table 8: The results show that the liquefiers of Examples 1 to 3 show obvious advantages in nucleic acid detection. Compared with treatment group 1A in Example 1, after omitting sodium carbonate in the liquefier (comparative group 3A) or replacing it with phosphate buffer (comparative group 4B), the Ct value of nucleic acid detection also lagged behind by about 3 Ct. This further proves that sodium carbonate mainly acts as a buffer, which can stabilize the pH value of the solution and prevent excessive alkalinity from damaging the sample. Moreover, its protection of the sample in this liquefier is better than that of phosphate buffer. After further increasing the concentration of strong base and buffer simultaneously (comparative example 3), the Ct value of nucleic acid detection also caused a certain impact. This shows that when too high a strong base liquefies the sample, it leads to the rapid release of pathogens, and the strong alkaline environment causes a certain loss of nucleic acid. Therefore, while meeting the liquefaction performance of the liquefier, it is also necessary to consider the protective effect on nucleic acid. At the same time, the same situation was shown with excessive sodium laurate (comparative group 1C). In addition, when isopropanol was replaced by ethanol and polyethylene glycol (Comparative Group 5B and Comparative Group 6B), or when a higher concentration of isopropanol was used (Comparative Example 5), more extreme results or complete inhibition of the PCR reaction occurred. At the same concentration, the inhibitory effect of isopropanol was less than that of the other two alcohol reagents (ethanol and polyethylene glycol).

[0072] Table 8 Streptococcus pneumoniae-Ct value

[0073]

[0074] In Table 8, “ / ” indicates that no signal value was detected and the result was negative.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection 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 solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A liquefier, characterized in that: The liquefier comprises a strong base, a carbonate, sodium laurate and isopropyl alcohol; in the liquefier, the concentration of the strong base is 0.5 mol / L to 4 mol / L, the concentration of the carbonate is 25 mmol / L to 200 mmol / L, the concentration of the sodium laurate is 20 mmol / L to 90 mmol / L, and the volume fraction of the isopropyl alcohol is 10% to 30%.

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

3. The liquefier according to claim 1, characterized in that The concentration of the carbonate is 50 mmol / L to 100 mmol / L.

4. The liquefier according to claim 1, characterized in that The concentration of the sodium laurate is 30 mmol / L to 60 mmol / L.

5. The liquefier according to claim 1, characterized in that The volume fraction of the isopropyl alcohol is 15% to 25%.

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

7. Use of the liquefaction agent 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. Use of the liquefaction agent 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 the liquefaction agent according to 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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