Probe combination, buffer solution, reagent combination, preparation method and activity detection method

Abstract

The invention provides a probe combination, a buffer solution, a reagent combination, a preparation method and an activity detection method, and relates to the technical field of biochemical detection. The probe combination can be used for simultaneously detecting DNase and RNase; comprising a DNA probe and an RNA probe, 5 ends of the DNA probe and the RNA probe are marked with fluorophores, and 3 ends of the DNA probe and the RNA probe are marked with quenching groups; the fluorophore comprises FAM and VIC; and the quenching group comprises BHQ1. The probe combination provided by the invention utilizes a fluorescence resonance energy transfer (FRET) principle, and realizes sensitive detection of DNase and RNase activity in one-time detection through FAM and VIC fluorophores and a BHQ1 quenching group on DNA and RNA probes. Fluorescence is weak when the probe is not cut by enzyme, fluorescence is enhanced after cutting, and enzyme activity is rapidly recognized. The method has the advantages of high sensitivity, high specificity and quick response, is suitable for various experimental environments, and improves the detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and in particular to a probe combination, a buffer solution, a reagent combination, a preparation method and an activity detection method. Background Art

[0002] Nucleases are a class of enzymes that can specifically hydrolyze the phosphodiester bonds of nucleic acid molecules. They are widely present in organisms and play a vital role in the metabolism and regulation of nucleic acids. Nucleases include two major categories: DNase and RNase. DNase specifically degrades DNA, while RNase is responsible for hydrolyzing RNA. These enzymes play an important role in the growth and development of organisms, gene expression regulation, and the occurrence and development of diseases. However, in some cases, improper expression or uncontrolled activity of nucleases may have adverse effects on organisms and even cause diseases. Therefore, it is particularly important to detect and control the activity of nucleases.

[0003] Among the methods for detecting nuclease activity, electrophoresis is a traditional technical means. This method indirectly reflects the activity of nucleases by observing the migration of nucleic acids during electrophoresis. Although electrophoresis has the advantages of simple operation and low cost, it has low sensitivity and is difficult to detect trace nuclease activity, and can only perform qualitative or semi-quantitative analysis. In addition, electrophoresis is time-consuming and not conducive to quickly obtaining test results, which is particularly inconvenient in experimental environments that require rapid response.

[0004] The radioactive labeling detection method is known for its high sensitivity. By labeling nucleic acid substrates with radioactive isotopes, the degradation process of nucleic acids can be accurately tracked. However, this method has obvious drawbacks, including radioactive hazards, high costs, and complex operations. The use of radioactive substances not only poses a threat to the health of experimenters, but also poses risks to environmental safety. In addition, radioactive labeled substrates and detection instruments are expensive, which limits their widespread application. The operation of radioactive labeling requires highly professional skills and strict operating procedures. The experimental process is cumbersome and prone to operating errors, which affects the reliability of the results.

[0005] Fluorescence detection is a detection method developed in recent years. It uses fluorescently labeled nucleic acid substrates to detect the activity of nucleases. This method has high sensitivity and can quickly detect enzyme activity, making it suitable for real-time monitoring and rapid screening. However, the specificity of fluorescence detection is poor. Non-enzyme factors such as pH changes and chemical interference may cause changes in fluorescence signals and produce false positive results. In addition, the presence of endogenous fluorescent substances in biological samples increases background interference and requires complex background correction. The stability of fluorescent labels is also a problem. The labels may fall off or quench during storage and experiments, affecting the reliability of the test results.

[0006] Colorimetry is a detection method based on color changes after enzyme-catalyzed reactions. This method is simple to operate, does not require complex instruments and equipment, is low in cost, and is suitable for preliminary screening of large-scale samples. However, the colorimetric method has low sensitivity and is insensitive to low-activity enzymes or trace residues. It may not be able to accurately detect small amounts of enzymes in the experimental environment or samples. In addition, the colorimetric method has poor accuracy, and color changes are easily affected by environmental factors (such as light, temperature) and sample matrix. It is difficult to accurately quantify the activity of the enzyme, and can only roughly judge the activity range.

[0007] In summary, existing nuclease activity detection methods have defects to varying degrees in terms of sensitivity, accuracy, ease of operation, safety, and cost. These defects limit the application of these methods in nuclease activity detection, especially in situations where rapid, accurate, and low-cost detection is required. Therefore, the development of a new, efficient, sensitive, safe, and low-cost nuclease activity detection method is of great significance for promoting nuclease-related research and its application in the biomedical field.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The first object of the present invention is to provide a probe combination, a buffer, a reagent combination, a preparation method and an activity detection method, wherein the probe combination utilizes the principle of fluorescence resonance energy transfer (FRET), and through the FAM and VIC fluorescent groups on the DNA and RNA probes and the BHQ1 quenching group, sensitive detection of DNase and RNase activity in one detection is achieved. When the probe is not cut by the enzyme, the fluorescence is weak, and after cutting, the fluorescence is enhanced, and the enzyme activity is quickly identified. This method has high sensitivity, high specificity, and rapid response, is applicable to a variety of experimental environments, and improves detection efficiency.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] In the first aspect, the present invention provides a probe combination, which can detect DNase and RNase simultaneously; the probe combination includes a DNA probe and an RNA probe; the 5' end of the DNA probe and the RNA probe are both labeled with a fluorescent group, and the 3' end is labeled with a quenching group; the fluorescent group includes FAM and VIC; the quenching group includes BHQ1. In an optional embodiment, the fluorescent group at the 5' end of the DNA probe is FAM, and the quenching group at the 3' end is BHQ1; in an optional embodiment, the fluorescent group at the 5' end of the RNA probe is VIC, and the quenching group at the 3' end is BHQ1. In an optional embodiment, the length of the DNA probe is 6 to 43 nucleotides; in an optional embodiment, the length of the DNA probe is 39 nucleotides; in an optional embodiment, the nucleotide sequence of the DNA probe is shown in SEQ ID NO.3.

[0012] In an optional embodiment, the length of the RNA probe is 8 to 20 nucleotides; in an optional embodiment, the length of the RNA probe is 16 nucleotides; in an optional embodiment, the nucleotide sequence of the RNA probe is shown in SEQ ID NO.16.

[0013] In a second aspect, the present invention provides a buffer solution, comprising: 10× reaction buffer solution, 2× freeze-drying protective agent and enzyme-free water. In an optional embodiment, the 10× reaction buffer solution comprises Tris-HCl, MgCl 2 , CaCl 2 and Tween-20; In an optional embodiment, the concentrations of the components in the 10× reaction buffer solution are: Tris-HCl 500 mM; MgCl 2 50mM; CaCl 2 is 25mM; Tween-20 1%; in an optional embodiment, the pH of the 10× reaction buffer solution is 7.5. In an optional embodiment, the 2× lyophilization protectant includes trehalose, PEG20000 and glycine; in an optional embodiment, the concentrations of the components in the 2× lyophilization protectant are: trehalose 6%; PEG20000 8%; glycine 1%.

[0014] In a third aspect, the present invention provides a reagent combination, comprising the probe combination as described in any one of the aforementioned embodiments, and the buffer as described in any one of the aforementioned embodiments.

[0015] In a fourth aspect, the present invention provides a method for preparing a reagent combination as described in the aforementioned embodiment, comprising: taking 1 μL of a DNA probe with a concentration of 5 μM and 1 μL of an RNA probe with a concentration of 5 μM in the probe combination, 2 μL of a 10× reaction buffer solution, 10 μL of a 2× freeze-drying protective agent, and 6 μL of enzyme-free water in a buffer to prepare a mixed reagent; sequentially performing a pre-freezing treatment, a sublimation drying treatment, and an analytical drying treatment on the mixed reagent to obtain the reagent combination;

[0016] In an optional embodiment, the temperature of the pre-freezing treatment is -60°C to -50°C; in an optional embodiment, the maintenance time of the pre-freezing treatment is 120 minutes; in an optional embodiment, the temperature of the sublimation drying treatment is -30°C; in an optional embodiment, the heating time of the sublimation drying treatment is 30 minutes; in an optional embodiment, the maintenance time of the sublimation drying treatment at -30°C is 600 minutes; in an optional embodiment, the temperature of the analytical drying treatment is 20°C; in an optional embodiment, the heating time of the analytical drying treatment is 120 minutes; in an optional embodiment, the maintenance time of the analytical drying treatment is 180 minutes.

[0017] In a fifth aspect, the present invention provides an activity detection method, based on the reagent combination described in the aforementioned embodiment, the activity detection method includes: taking a target sample, adding it to a reaction container containing a probe combination and a buffer solution in the reagent combination, to obtain a mixed sample to be tested; detecting the mixed sample to be tested using a fluorescent quantitative PCR device to obtain a fluorescence curve; determining whether DNase and RNase are present in the target sample based on the fluorescence curve; in an optional embodiment, ensuring that the total amount of the mixed sample to be tested in the reaction container is 20 μL; wherein the insufficient part is supplemented with enzyme-free water; in an optional embodiment, the detection channels of the fluorescent quantitative PCR device select FAM channel and VIC channel; in an optional embodiment, during the detection of the mixed sample to be tested using a fluorescent quantitative PCR device, the reaction conditions of the fluorescent quantitative PCR device include: reaction temperature 37°C; reaction time is at least 30 minutes.

[0018] In an optional embodiment, determining whether DNase and RNase exist in the target sample according to the fluorescence curve includes: if there is a fluorescence curve signal in the FAM channel, determining that DNase exists in the target sample; if there is a fluorescence curve signal in the VIC channel, determining that RNase exists in the target sample.

[0019] The probe combination of the present invention realizes sensitive detection of DNase and RNase activity in a sample by utilizing the principle of fluorescence energy transfer (FRET). The probe combination includes a DNA probe and an RNA probe labeled with fluorescent groups FAM and VIC, and a quenching group BHQ1. When not cut by an enzyme, no fluorescent signal is generated because the fluorescent group and the quenching group are close to each other. When the sample contains DNase or RNase activity, the probe is degraded, and the fluorescent group and the quenching group are separated from each other, thereby generating a gradually enhanced fluorescent signal. This design allows to simultaneously determine whether a sample contains DNase and RNase through a single detection, thereby improving the efficiency and practicality of the detection. Through the fluorescence increase value of FAM and VIC, the residual enzyme activity of DNase / RNase in the sample can be sensitively detected. In addition, the probe combination has high sensitivity, high specificity, rapid response and a wide range of applications, which makes it have significant advantages in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The figure is the result of DNA probe screening in Example 1 (SEQ ID NO.1-4);

[0022] Figure 2 The figure is the result of DNA probe screening in Example 1 (SEQ ID NO.5-8);

[0023] Figure 3 The figure is the result of DNA probe screening in Example 1 (SEQ ID NO.9-12);

[0024] Figure 4 The figure is the result of RNA probe screening in Example 1 (SEQ ID NO.13-16);

[0025] Figure 5 The figure is the result of RNA probe screening in Example 1 (SEQ ID NO.17-19);

[0026] Figure 6 This is the result diagram of Buffer2 and FAM-DNA+VIC-RNA modification method in Example 2;

[0027] Figure 7 The specificity result diagram in Example 3 (reagents A to B);

[0028] Figure 8 The specificity result diagram in Example 3 (reagents C to D);

[0029] Fig. 9 Figure (①) is the screening result of DNase lyophilization protectant in Example 4;

[0030] Fig.10 The figure (②) is the screening result of DNase lyophilization protectant in Example 4;

[0031] Fig.11 The figure (③) is the screening result of DNase lyophilization protectant in Example 4;

[0032] Fig.12 The figure (④) is the screening result of DNase freeze-drying protective agent in Example 4;

[0033] Fig.13 The RNase freeze-drying protective agent screening results in Example 4 (①~④);

[0034] Fig.14 DNase detection limit result diagram in Example 5;

[0035] Fig.15 Result diagram of RNase detection limit in Example 5;

[0036] Fig.16 This is a graph showing the detection results of the target sample in Example 6. DETAILED DESCRIPTION

[0037] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0038] In an embodiment of the present application, a probe combination is provided, which can detect DNase and RNase simultaneously; the probe combination includes a DNA probe and an RNA probe; the 5' end of the DNA probe and the RNA probe are both labeled with a fluorescent group, and the 3' end is labeled with a quenching group. The fluorescent group includes FAM and VIC; the quenching group includes BHQ1.

[0039] The probe combination in the present application is intended to simultaneously detect the activities of two nucleases, DNase and RNase. The probe combination includes a DNA probe and an RNA probe labeled with fluorescent groups FAM and VIC, and a quenching group BHQ1.

[0040] As mentioned above, the fluorophores FAM and VIC were chosen because they have spectrally distinct properties and can generate distinguishable fluorescent signals, thus allowing for the simultaneous detection of two different enzyme activities.

[0041] As mentioned above, the quencher group BHQ1 was selected because it has a highly efficient quenching effect on the fluorescence of FAM and VIC. When the probe is not cleaved by the enzyme, the distance between the fluorescent group and the quencher group is relatively close, and no or very weak fluorescence signals are generated.

[0042] It should be noted that the significance of simultaneous detection of DNase and RNase is that these two enzymes play key roles in many biological processes, such as DNA replication, RNA processing and apoptosis. Simultaneous detection of the activities of these two enzymes can help researchers understand the biological status of the sample and the changes in enzyme activity more comprehensively.

[0043] As described above, constructing a combination of DNA probes and RNA probes can provide specific detection for different types of nucleases, because DNA and RNA have different structures and stabilities and different sensitivities to specific enzymes. This combination can improve the sensitivity and specificity of the detection, because any single probe type may not provide enough information to distinguish and quantify the two enzyme activities.

[0044] Based on the above principles, the probe combination provided in this embodiment has the following beneficial effects:

[0045] (1) Fluorescence energy transfer (FRET) mechanism: Fluorescence energy transfer is achieved by utilizing the spectral overlap between the BHQ1 quencher and the FAM / VIC fluorescent group. When the probe is not cleaved by the enzyme, the fluorescence energy emitted by the fluorescent group is absorbed by the quencher, resulting in a decrease in fluorescence intensity, which helps to reduce background signals and improve detection specificity.

[0046] (2) Highly sensitive detection: Due to the close distance between the FAM / VIC fluorescent group and the BHQ1 quenching group, any slight change (such as probe breakage caused by enzyme cleavage) will lead to a significant change in fluorescence intensity, which enables the probe combination to detect very low levels of enzyme activity.

[0047] (3) Rapid response: After the probe is cut, the fluorescent group and the quenching group separate rapidly and the fluorescence signal is immediately enhanced, which allows the experiment to be completed in a short time (e.g., within 30 minutes) and speeds up the detection process.

[0048] (4) High specificity: Since the generation of fluorescent signals depends on the cleavage of specific enzymes, this probe combination can distinguish specific types of enzyme activity, thereby improving the specificity of detection.

[0049] (5) Simplified experimental design: Since the probe combination can detect two enzymes (DNase and RNase) simultaneously, the experimental design is simplified, reducing the number of experiments required and the sample processing steps.

[0050] (6) Wide applicability: This probe combination can be applied to a variety of different experimental environments and sample types, including clinical samples and environmental monitoring, and has wide applicability.

[0051] (7) Easy to operate: The design of the probe combination makes the operation process simple and easy to master, reducing the difficulty and error rate of experimental operations.

[0052] (8) Cost-effectiveness: Since the design of the probe combination allows the simultaneous detection of multiple enzymes in the same experiment, this reduces the number and cost of probes required for the experiment and improves the economy of the experiment.

[0053] In summary, the probe combination in the embodiments of the present application provides an efficient, sensitive, rapid and specific method for detecting enzyme activity by utilizing the fluorescence energy transfer mechanism, which has significant advantages and broad application prospects.

[0054] In some embodiments, the fluorescent group at the 5' end of the DNA probe is FAM, and the quenching group at the 3' end is BHQ1. In some embodiments, the fluorescent group at the 5' end of the RNA probe is VIC, and the quenching group at the 3' end is BHQ1. In some embodiments, the length of the DNA probe is 6 to 43 nucleotides; in some embodiments, the length of the DNA probe is 39 nucleotides. In some embodiments, the nucleotide sequence of the DNA probe is shown in SEQ ID NO.3.

[0055] The nucleotide sequence of the DNA probe is shown in SEQ ID NO. 3, and can be specifically shown in Table 1 below.

[0056] In some embodiments, the RNA probe has a length of 8 to 20 nucleotides. In some embodiments, the RNA probe has a length of 16 nucleotides. In some embodiments, the nucleotide sequence of the RNA probe is shown in SEQ ID NO.16.

[0057] The nucleotide sequence of the RNA probe is shown in SEQ ID NO.16, and can be specifically shown in Table 1 below.

[0058] Table 1. Nucleotide sequences of DNA probes and RNA probes

[0059]

[0060] In a second aspect, the present invention provides a buffer solution, comprising: 10× reaction buffer solution, 2× freeze-dried protective agent and enzyme-free water. The 10× reaction buffer solution comprises Tris-HCl, MgCl 2 , CaCl 2 and Tween-20.

[0061] In some embodiments, the 10× DNA Reaction Buffer solution includes: Tris-HCl, MgCl 2 , CaCl 2 and Tween-20.

[0062] In some embodiments, the concentrations of the components in the 10×DNA Reaction Buffer solution are as shown in Table 2.

[0063] Table 2. Concentration of components in 10×DNA Reaction Buffer solution

[0064] Components Tris-HCl <![CDATA[MgCl 2 ]]> <![CDATA[CaCl 2 ]]> Tween-20 concentration 500mM 50mM 25mM 1％

[0065] In some embodiments, the pH of the 10× reaction buffer solution is 7.5.

[0066] In some embodiments, the 2× lyoprotectant comprises trehalose, PEG20000 and glycine.

[0067] In some embodiments, the components of the 2× lyophilized protective agent are as shown in Table 3.

[0068] Table 3. Concentration of each component in 2× lyophilized protective agent

[0069] Components Trehalose PEG20000 Glycine

[0070] A reagent combination is also provided in an example of the present application, comprising the probe combination as described in any one of the aforementioned embodiments, and the buffer as described in any one of the aforementioned embodiments.

[0071] The above-mentioned reagent combination can constitute a product for activity detection, for example, a kit.

[0072] The present application also provides a method for preparing the reagent combination as described in the above embodiment, comprising:

[0073] Step S1, 1 μL of a 5 μM DNA probe and 1 μL of a 5 μM RNA probe in the probe combination, 2 μL of a 10× reaction buffer solution, 10 μL of a 2× freeze-dried protective agent, and 6 μL of enzyme-free water in the buffer are prepared to obtain a mixed reagent, as shown in Table 4.

[0074] Table 4. Components and corresponding amounts in mixed reagents

[0075] Components DNA probe RNA probes 10× Reaction Buffer 2× Lyoprotectant Enzyme-free water Add μL per reaction 1 1 2 10 6

[0076] Step S2, sequentially performing pre-freezing treatment, sublimation drying treatment and analytical drying treatment on the mixed reagent to obtain the reagent combination.

[0077] In the examples of the present application, the reagent combination provided is a freeze-dried reagent combination product. The above preparation method is a preparation method for preparing various reagents in the reagent combination together and performing freeze-drying treatment. The freeze-dried reagent combination (a mixture of multiple reagents and substances) provided in the examples of the present application is simpler to operate than the liquid reagents in conventional technology, and does not require reagent preparation steps. It is only necessary to add the sample to the freeze-dried reagent combination product to conduct the experiment; at the same time, the freeze-dried reagent combination is more stable in storage.

[0078] As mentioned above, the reason why the reagent combination is freeze-dried is to improve the stability and shelf life of the reagent. Lyophilization is a technology that removes water from a solution through a freezing and vacuum drying process to obtain a dry powder or granular substance.

[0079] As mentioned above, this process includes the following key steps: First, in the first step of the freeze-drying process, the mixed reagents are cooled below their freezing point, causing the water in them to freeze. The purpose of pre-freezing is to ensure that during the subsequent sublimation drying process, the water can directly change from solid to gas without passing through the liquid state. This helps to avoid the formation of a liquid layer during the drying process, which may cover the surface of the sample and hinder the further evaporation of water. Then, after pre-freezing, the system will apply a vacuum and gradually increase the temperature so that the ice crystals sublime directly from the solid state to water vapor, a process called sublimation. The sublimation drying process is carried out at low temperatures, which helps to maintain the stability of the sample and prevent sample degradation caused by high temperatures.

[0080] After sublimation drying, there may still be a small amount of unfrozen water in the sample. The desorption drying process is performed at a slightly higher temperature to remove this residual water. This step ensures that the final moisture content of the reagent is extremely low, thereby improving the long-term stability and shelf life of the reagent.

[0081] Freeze-drying is a method of removing water from reagents through a freezing and vacuum drying process, which can significantly improve the stability of the reagents and reduce their sensitivity to heat and humidity, allowing them to be stably stored under a wider range of conditions. In addition, freeze-drying effectively reduces the rate of chemical reactions by reducing the moisture content in the reagents, helping to extend the shelf life of the reagents. Since freeze-dried reagents are usually in the form of dry powders or granules, this is not only convenient for transportation and storage because they occupy less space, but also reduces degradation and contamination that may occur during transportation and storage, making the reagents more convenient to use in experiments.

[0082] In some embodiments, the temperature of the pre-freezing treatment is -60°C to -50°C; for example, the temperature can be -60°C, -58°C, -55°C, -53°C, -50°C, etc. In some embodiments, the pre-freezing treatment is maintained for 120 minutes; in some embodiments, the temperature of the sublimation drying treatment is -30°C; in some embodiments, the heating time of the sublimation drying treatment is 30 minutes; in some embodiments, the maintenance time of the sublimation drying treatment at -30°C is 600 minutes; in some embodiments, the temperature of the desorption drying treatment is 20°C; in some embodiments, the heating time of the desorption drying treatment is 120 minutes; in some embodiments, the maintenance time of the desorption drying treatment is 180 minutes.

[0083] The present application also provides an activity detection method in an embodiment, based on the reagent combination described in the above embodiment, or the reagent combination prepared by the preparation method described in the above embodiment, the activity detection method comprises:

[0084] Step S10, taking a target sample and adding it into a reaction container containing the probe combination in the reagent combination and a buffer solution to obtain a mixed sample to be tested.

[0085] As mentioned above, the "target sample" refers to a sample to be tested that may contain DNase (deoxyribonuclease) and / or RNase (ribonuclease) biochemical activity. The sample is used to confirm whether the sample is contaminated by these enzymes, or for quality control to ensure the integrity of the sample, especially in scenarios involving nucleic acid extraction and subsequent experimental operations.

[0086] These samples can be any form of biological samples, such as cell lysates, tissue extracts, purified enzyme preparations, environmental samples (such as nucleases in water and soil samples), or any other biological or environmental samples that may contain DNase and RNase.

[0087] As mentioned above, the target sample is added to the reaction vessel containing the reagent combination to obtain a mixed sample to be tested. If the total reaction volume is less than 20 μL, it is supplemented to 20 μL with enzyme-free water to obtain a mixed solution containing the probe and the target sample, which will be used for subsequent fluorescence quantitative detection.

[0088] This step ensures that the reaction system has sufficient volume for accurate fluorescence detection, and the use of enzyme-free water avoids additional interference from enzyme activity.

[0089] Step S20, detecting the mixed sample to be tested by using a fluorescence quantitative device to obtain a fluorescence curve.

[0090] In the above, the mixed sample to be tested is detected by using a fluorescence quantitative device to obtain a fluorescence curve. The fluorescence intensity change of a specific channel (FAM and VIC) can be observed through the fluorescence curve, thereby determining whether DNase and RNase exist in the target sample.

[0091] A special fluorescence quantitative instrument can be used to set appropriate detection parameters, such as temperature, time, etc. For example, a fluorescence quantitative PCR instrument, such as ABI7500 fluorescence quantitative PCR instrument, can be used.

[0092] Step S30, determining whether DNase and RNase exist in the target sample according to the fluorescence curve.

[0093] As described above, the presence of DNase and RNase in the target sample is determined based on the fluorescence curve. If a fluorescence curve appears in the FAM channel, it indicates that DNase exists in the sample; if a fluorescence curve appears in the VIC channel, it indicates that RNase exists in the sample.

[0094] In addition, if no fluorescence curve appears in the FAM channel, it can be determined that there is no DNase in the target sample; if no fluorescence curve appears in the VIC channel, it can be determined that there is no RNase in the target sample. By detecting dual-channel fluorescence signals, it is possible to simultaneously determine whether the sample contains DNase and RNase, thereby improving the efficiency and accuracy of the detection. Specifically, the data output by the fluorescence quantification device is analyzed to identify changes in the fluorescence signal of a specific channel.

[0095] In this method, the design of DNA probes and RNA probes and the strategy of fluorescent labeling work together to achieve sensitive detection of DNase and RNase activity. The FAM and VIC fluorescent groups labeled at the 5' end of the DNA probe and RNA probe, as well as the BHQ1 quenching group at the 3' end, use the principle of fluorescence resonance energy transfer (FRET). When not cut, the fluorescent group and the quenching group are close to each other, so the fluorescence signal is weak; when the probe is cut by the corresponding enzyme, the fluorescent group and the quenching group are separated, and the fluorescence signal is enhanced. This design enables this method to detect the activity of the target enzyme with high sensitivity, and through a single detection, information on whether the sample contains DNase and RNase can be obtained at the same time, which has the advantages of simple operation, rapidity, high sensitivity and high specificity.

[0096] In some embodiments, the total amount of the mixed sample to be tested in the reaction container is ensured to be 20 μL; wherein the insufficient amount is supplemented with enzyme-free water;

[0097] In some embodiments, the detection channels of the fluorescence quantitative PCR device are selected from FAM channel and VIC channel; in some embodiments, during the detection of the mixed sample to be tested by the fluorescence quantitative PCR device, the reaction conditions of the fluorescence quantitative PCR device include: reaction temperature of 37°C; reaction time of at least 30 minutes. Increasing the reaction time can improve the detection sensitivity.

[0098] The step S30, determining whether DNase and RNase exist in the target sample according to the fluorescence curve, comprises: step S31, if there is a fluorescence curve signal in the FAM channel, determining that DNase exists in the target sample; step S32, if there is a fluorescence curve signal in the VIC channel, determining that RNase exists in the target sample.

[0099] In the embodiments of the present application, a specific judgment method is provided, and the results are judged by means of a dual-channel fluorescence curve. For the same sample, only one operation is required to obtain two results, namely, whether there is DNase or RNase contamination, which greatly increases the throughput, saves reagent costs, and makes the operation easier.

[0100] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0101] Relevant information of reagents, materials and instruments used in the examples:

[0102] Table 5. Reagent information

[0103] Reagent name Reagent Part Number Manufacturer Trehalose A422870 Bioengineering Glycine A110167 Bioengineering PEG20000 A601790 Bioengineering 1M Tris-HCl, pH 7.5 B548124 Bioengineering <![CDATA[MgCl 2 ]]> A601336 Bioengineering <![CDATA[CaCl 2 ]]> A501330 Bioengineering Tween-20 A600560 Bioengineering DNaseⅠ B618252 Bioengineering RNase A A002131 Bioengineering

[0104] Table 6. Material information

[0105] Material Name Material Part Number Manufacturer Fluorescence quantitative PCR 8-tube strip & cap F603001-0001 Bioengineering

[0106] Table 7. Experimental instrument information

[0107] Instrument Name Instrument Model Manufacturer qPCR ABI7500 Thermo Fisher Scientific Freeze Dryer Polit2-4M Bo Yi Kang

[0108] Example 1: Design of probe combination

[0109] DNase can be divided into endonucleases and exonucleases. Endonucleases cut phosphodiester bonds inside the DNA chain, such as DNase I, which can randomly hydrolyze phosphodiester bonds inside double-stranded or single-stranded DNA. Usually divalent metal ions are required as cofactors to cut DNA into oligonucleotide fragments under appropriate pH and temperature conditions, producing fragments with 5'-phosphate ends and 3'-hydroxyl ends. Exonucleases cut nucleotides one by one from the ends of the DNA chain.

[0110] Ribonuclease A (RNase A) is derived from bovine pancreas and is an endoribonuclease that can specifically attack the 3' end of pyrimidine (C, U) residues on RNA, cleaving the phosphodiester bond formed by cytosine or uracil and the adjacent nucleotide. The final products of the reaction are 3' pyrimidine nucleotides and oligonucleotides with 3' pyrimidine nucleotides at the end.

[0111] RNase T1 is an endoribonuclease that specifically acts on the 3' phosphate of guanine (G) nucleotides and cuts the phosphodiester bond connecting the adjacent nucleotides. The end products of the reaction are guanosine 3' phosphate and oligonucleotides with guanosine 3' phosphate at the end.

[0112] According to the above-mentioned DNase characteristics, natural and artificial sequences were designed respectively, and to ensure its background fluorescence, linear probes and molecular beacon probes were designed respectively.

[0113] At the same time, in view of the above-mentioned RNase characteristics, a fully folded complementary probe was designed to ensure its background fluorescence, and a straight-chain probe composed of UG was designed to ensure its sensitivity.

[0114] The specific designed probes and sequences are as follows:

[0115] Table 8. DNA probes to be screened and corresponding sequences

[0116]

[0117] Table 9. RNA probes to be screened and corresponding sequences

[0118] SEQ ID NO. Fluorophore Nucleotide sequence Quenching group Attached photos 13 VIC GCTACCTATGGTAGC BHQ1 13 14 VIC TGTGTGTGTGTGTGTGTGTG BHQ1 14 15 VIC TACCTATGGTA BHQ1 15 16 VIC TGTGTGTGTGTGTGTG BHQ1 16 17 VIC TGTGTGTGTGTG BHQ1 17 18 VIC TGTGTGTG BHQ1 18 19 VIC TGTGTGTGTGCACACA BHQ1 19

[0119] Screening method:

[0120] (1) DNA probe screening: 0.0005U / T DNase was tested at the same amount for the above DNA probes, and enzyme-free water was used as a negative control. According to 1μL of DNA probe (5μM), 2μL of 10× reaction buffer, 1μL of 0.0005U / μL DNase was added, and the reaction system was made up to 20μL with enzyme-free water. The reaction was carried out on an ABI fluorescent quantitative PCR instrument 7500 at 37℃ for 1min / cycle and 30 cycles.

[0121] (2) RNA probe screening: 0.3 pg / T RNase was tested at the same amount for the above RNA probes, and enzyme-free water was used as a negative control. According to 1 μL of RNA probe (5 μM), 2 μL of 10× reaction buffer, 1 μL of 0.3 pg / T RNase was added, and the reaction system was made up to 20 μL with enzyme-free water. The reaction was carried out on an ABI fluorescent quantitative PCR instrument 7500 at 37°C for 1 min / cycle and 30 cycles.

[0122] Experimental results:

[0123] (1) DNA probe screening results: Figure 1 to Figure 3 As shown, SEQ ID NO. 1 to 6 can detect 0.0005 U / T, among which SEQ ID NO. 3 has the highest fluorescence value and the best repeatability, so NO. 3 is selected as the preferred DNA probe of the present invention.

[0124] (2) RNA probe screening results: Figure 4-5 As shown, SEQ ID NO.16 has the highest fluorescence value, so NO.16 is selected as the preferred RNA probe of the present invention.

[0125] In summary, according to the experimental results, in the final preferred probe combination in this embodiment, the sequences of the DNA probe and the RNA probe are as follows: (1) DNA probe SEQ ID NO.3: [5`6-FAM]-CCACAGCCAAGGAGCGCCTCATCCTGGCCTTGGCTGTGG-[3`BHQ1]; (2) RNA probe SEQ ID NO.16: [5`VIC]-TGTGTGTGTGTGTGTG-[3`BHQ1].

[0126] Example 2: Investigation of reaction system and probe modification groups

[0127] Experimental methods:

[0128] Table 10. Components of three 10× reaction buffers

[0129] Components Buffer1 Buffer2 Buffer3 Components Buffer1 Buffer2 Buffer3 Tris-HCl 500mM 500mM 500mM <![CDATA[CaCl 2 ]]> 10mM 25mM 50mM <![CDATA[MgCl 2 ]]> 50mM 50mM 50mM Tween-20 1％ 1％ 1％

[0130] (1) First, according to Table 10, three 10× reaction buffer solutions were prepared, and two labeled DNA probes and RNA probes (FAM and VIC) were synthesized for each. (2) Then, the reaction system was prepared according to 2μL 10× reaction buffer solution, 1μL DNA probe (5μM), 1μL RNA probe (5μM), 2μL DNase, 2μL RNase and 12μL enzyme-free water. (3) For each combination of 10× reaction buffer solutions (Buffer1~Buffer3), the modification combination of the two probe fluorescent groups was matched (see the table for specific grouping), respectively: a, DNA probe labeled FAM + RNA probe labeled VIC; b, DNA probe labeled VIC + RNA probe labeled FAM. (4) The detection effects of 0.001U / T DNase and 1pg / T RNase under different reaction buffer and probe modification combinations were tested respectively, and enzyme-free water was used as a negative control. The reaction was carried out on an ABI fluorescent quantitative PCR instrument 7500 at 37℃ for 1min / cycle and 30 cycles. Please refer to Table 11 and Table 12 for the experimental results.

[0131] Table 11. Experimental results (ABC)

[0132]

[0133] Table 12. Experimental results (DEF)

[0134]

[0135] The results are shown in Tables 11 and 12. For the same labeling method, the fluorescence value of the positive reaction of Buffer 2 is the highest, and the negative fluorescence value is the lowest. Under Buffer 2, the positive fluorescence value of FAM-DNA+VIC-RNA is high and the negative fluorescence value is low. Finally, Buffer 2 was selected as the preferred 10× reaction buffer of the present invention, and the FAM-DNA+VIC-RNA modification method was used as the probe modification scheme of the present invention. The results are shown in FIG. Figure 6 .

[0136] Example 3: Investigation of the specificity of probe combinations

[0137] Experimental method: To determine the specificity of the two probes, the reaction system was prepared according to Table 13 below, and 0.001U / T DNase and 1pg / T RNase were tested respectively. Enzyme-free water was used as a negative control. The reaction was carried out on an ABI fluorescent quantitative PCR instrument 7500 at 37°C for 1 min / cycle and for 30 cycles.

[0138] Table 13. Reaction system table (unit: μL)

[0139] Reagents A B C D Reagents A B C D 10× Buffer Solution 2 2 2 2 DNase I 2 0 2 0 DNase-Probe (5 μM) 1 1 1 1 RNase A 2 2 0 0 RNase-Probe (5 μM) 1 1 1 1 <![CDATA[ddH 2 The]]> 12 14 14 16

[0140] Experimental results: The results are as follows Figure 7-Figure 8 As shown, both DNA probes and RNA probes have good specificity and no cross-reaction.

[0141] Example 4: Investigation of freeze-dried protective agent formulation

[0142] Experimental method: (1) Prepare 4 kinds of 2× lyophilization protective agents: ① 6% trehalose and 1% glycine; ② 8% PEG20000; ③ 6% PEG20000; ④ 6% trehalose, 1% glycine and 8% PEG20000.

[0143] (2) Prepare the reaction system by adding 2 μL 10× reaction buffer, 10 μL 2× lyophilization buffer, 1 μL DNA probe (5 μM), 1 μL RNA probe (5 μM), 2 μL DNase, 2 μL RNase, and 2 μL enzyme-free water to make up to 20 μL.

[0144] (3) 0.0005 U / T DNase and 0.1 pg / T RNase were tested respectively, and enzyme-free water was used as a negative control. The reaction was carried out on an ABI fluorescent quantitative PCR instrument 7500 at 37°C for 1 min / cycle and for 30 cycles.

[0145] Experimental results: The results are as follows Figures 9 to 12 As shown, the DNase results showed that there were no false positives in the four groups, among which the fluorescence value of No. ④ 2× lyophilized protective agent was the highest. Fig.13 The results showed that RNase ① and ④ had no false positives, but the fluorescence value of ④ was higher. Therefore, formulation ④ was finally selected as the 2× lyophilization protectant.

[0146] Example 5: Sensitivity Study

[0147] Experimental method: (1) First, add 10 μL of the above 2× lyophilized protective agent to every 20 μL reaction system, then add 2 μL of 10× reaction buffer, 1 μL of DNA probe (5 μM), 1 μL of RNA probe (5 μM) and 6 μL of enzyme-free water to prepare the lyophilized reagent.

[0148] (2) Freeze drying is performed as follows: Before preparing the freeze-dried reagent, open the freeze dryer and precool it to -55°C. After the reagent is prepared, place the metal ice plate containing the reagent on the freeze dryer plate layer, close the box, prefreeze at -55°C and start freeze drying after 60 minutes based on the existing time. The freeze drying program settings are shown in Table 14.

[0149] Table 14. Freeze-drying procedure

[0150]

[0151] After the freeze-drying is completed, turn on the laboratory dehumidifier first, and when the humidity drops below 20%, start collecting the freeze-dried reagents. After the eight-tube strips are covered, they are divided into aluminum foil bags, and desiccant is added. Vacuum, seal, and store at -20°C.

[0152] (3) Then, DNase I (1 U / μL) was diluted to 1×10 -3 / 1×10 -4 / 2×10 -5 / 1×10 -5 U / μL, RNaseA (10 mg / mL) was diluted to 0.6 / 0.3 / 0.1 / 0.05 pg / μL with enzyme-free water, and the mixture was prepared into 1 μL diluted DNase / RNase + 19 μL water;

[0153] (4) Take 20 μL of each sample and add it to the freeze-dried system. Incubate the reaction on an ABI fluorescent quantitative PCR instrument 7500 at 37°C for 1 min / cycle for 30 cycles.

[0154] Experimental results: The results are as follows Fig.14 As shown in the figure, the minimum detection limit of the kit for DNase is 1×10 -5 U / T, Fig.15 The results showed that the minimum detection limit of the kit for RNase was 0.05pg / T.

[0155] Example 6: Activity Detection

[0156] Experimental method: Set 4 real samples as target samples, namely:

[0157] Desktop swabs, BSA reagent from a certain manufacturer, 1.5mL centrifuge tubes from a certain manufacturer, and wastewater from a laboratory sink.

[0158] (1) First, the sample was processed by soaking a desktop swab in 500 μL of enzyme-free water; dissolving 0.1 mg of BSA in 500 μL of enzyme-free water; repeatedly rinsing 10 1.5 mL centrifuge tubes with 500 μL of enzyme-free water; and taking 100 μL of sewage from the laboratory pool.

[0159] (2) Then, take 20 μL of each sample and add it to the reaction tube of the lyophilized reagent combination;

[0160] (3) Vortex to mix, and spin for a few seconds;

[0161] (4) Place the tube on an ABI fluorescent quantitative PCR instrument 7500, select FAM and VIC as the fluorescence channels, and react at 37°C for 1 min / cycle for 30 cycles.

[0162] Experimental results: Observe the fluorescence curve, the results are as follows Fig.16 As shown, RNase was detected in the desktop swab, which was weakly positive; RNase was detected in the BSA, which was strongly positive and heavily contaminated; DNase and RNase were contaminated in the sewage in the laboratory pool; and no 1.5mL centrifuge tube was detected.

[0163] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probe combination, characterized in that: The probe combination can detect DNase and RNase simultaneously; the probe combination includes a DNA probe and an RNA probe; The 5' end of the DNA probe and the RNA probe are both labeled with a fluorescent group, and the 3' end is labeled with a quenching group; The fluorescent group includes FAM and VIC; the quenching group includes BHQ1; Preferably, the fluorescent group at the 5' end of the DNA probe is FAM, and the quenching group at the 3' end is BHQ1; Preferably, the fluorescent group at the 5' end of the RNA probe is VIC, and the quenching group at the 3' end is BHQ1.

2. The probe combination according to claim 1, characterized in that: The length of the DNA probe is 6 to 43 nucleotides; Preferably, the length of the DNA probe is 39 nucleotides; Preferably, the nucleotide sequence of the DNA probe is shown as SEQ ID NO.

3.

3. The probe combination according to claim 1, characterized in that: The length of the RNA probe is 8 to 20 nucleotides; Preferably, the RNA probe is 16 nucleotides in length; Preferably, the nucleotide sequence of the RNA probe is shown as SEQ ID NO.

16.

4. A buffer solution, characterized in that include: 10× reaction buffer solution, 2× lyophilization protectant and enzyme-free water.

5. The buffer according to claim 4, characterized in that The 10× reaction buffer solution includes Tris-HCl, MgCl2, CaCl2 and Tween-20; Preferably, the concentrations of the components in the 10× reaction buffer solution are: Tris-HCl 500 mM; MgCl2 50mM; CaCl2 is 25 mM; Tween-20 1%; Preferably, the pH of the 10× reaction buffer solution is 7.

5.

6. The buffer according to claim 4, characterized in that The 2× lyophilization protective agent includes trehalose, PEG20000 and glycine; Preferably, the concentrations of the components in the 2× lyophilized protective agent are: Trehalose 6%; PEG20000 8%; Glycine 1%.

7. A reagent combination, characterized in that: The method comprises the probe combination according to any one of claims 1 to 3, and the buffer according to any one of claims 4 to 6.

8. A method for preparing the reagent combination as claimed in claim 7, characterized in that: include: Take 1 μL of a 5 μM DNA probe and 1 μL of a 5 μM RNA probe in the probe combination, 2 μL of a 10× reaction buffer solution, 10 μL of a 2× freeze-dried protective agent, and 6 μL of enzyme-free water in the buffer to prepare a mixed reagent; The mixed reagent is sequentially subjected to pre-freezing treatment, sublimation drying treatment and analytical drying treatment to obtain the reagent combination; Preferably, the temperature of the pre-freezing treatment is -60°C to -50°C; Preferably, the pre-freezing treatment is maintained for 120 minutes; Preferably, the temperature of the sublimation drying treatment is -30°C; Preferably, the heating time of the sublimation drying process is 30 minutes; Preferably, the maintenance time at -30°C during the sublimation drying process is 600 minutes; Preferably, the temperature of the analytical drying process is 20°C; Preferably, the heating time of the analytical drying process is 120 minutes; Preferably, the analytical drying process is maintained for 180 minutes.

9. An activity detection method based on the reagent combination of claim 7, characterized in that: The activity detection method comprises: Taking a target sample and adding it to a reaction container containing the probe combination and a buffer in the reagent combination to obtain a mixed sample to be tested; The mixed sample to be tested is tested using a fluorescent quantitative PCR device to obtain a fluorescence curve; Determine whether DNase and RNase exist in the target sample according to the fluorescence curve; Preferably, the total amount of the mixed sample to be tested in the reaction container is ensured to be 20 μL; wherein the insufficient amount is supplemented with enzyme-free water; Preferably, the detection channels of the fluorescent quantitative PCR device are selected from FAM channel and VIC channel; Preferably, when the mixed sample to be tested is tested using a fluorescent quantitative PCR device, the reaction conditions of the fluorescent quantitative PCR device include: Reaction temperature 37°C; The reaction time is at least 30 minutes.

10. The activity detection method according to claim 9, characterized in that: Determining whether DNase and RNase exist in the target sample according to the fluorescence curve comprises: If there is a fluorescence curve signal in the FAM channel, it is determined that DNase exists in the target sample; If there is a fluorescence curve signal in the VIC channel, it is determined that RNase exists in the target sample.

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