Fluorescent probe for detecting DNA enzyme single-chain hydrolytic activity and application thereof

Abstract

The invention provides a fluorescent probe for detecting DNA enzyme single-chain hydrolytic activity and application of the fluorescent probe. The fluorescent probe is of a single-stranded DNA structure and contains a fluorophore and a quenching group, and the distance between the fluorophore and the quenching group is 5-15 bp. The fluorescent probe provided by the invention is obtained by simultaneously inserting a fluorophore and a quenching group on the single-stranded DNA and controlling the distance between the fluorophore and the quenching group within the range of 5-15 bp, and the fluorescent probe is applied to detection of the DNA enzyme single-stranded hydrolytic activity. By detecting the fluorescence signal in the detection system, the purpose of detecting the DNA enzyme single-chain hydrolytic activity in the sample to be detected can be achieved. The fluorescent probe provided by the invention can be used for carrying out specific detection on the DNA enzyme single-chain hydrolytic activity, the detection sensitivity is high, the detection ground noise is relatively low, the experimental error is reduced, and the accuracy of the detection result is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a fluorescent probe for detecting DNA enzyme single-strand hydrolysis activity and an application thereof. Background Art

[0002] DNA enzyme, also known as deoxyribonuclease (DNase), is widely present in cells and is involved in a variety of physiological and biochemical processes, such as DNA replication and repair, cell apoptosis, etc. It can also be used as a disease marker. For example, the expression level of DNase I is low in patients with arthritis and nephritis. It can also be used to treat diseases, such as using DNase I to treat cystic fibrosis.

[0003] DNA enzymes mainly cut various DNA chains by catalyzing the phosphodiester bonds in the main chain of DNA molecules. They are important participants in biological processes and are also an important class of hydrolases for studying biological mechanisms.

[0004] In recent years, the most studied DNA enzyme is DNA enzyme I (DNase I). DNase I can hydrolyze DNA to produce mononucleotides and oligodeoxyribonucleotides with 5'-phosphate and 3'-hydroxyl groups. The hydrolysis activity of DNase I depends on calcium ions and can be activated by magnesium ions or divalent manganese ions. In the presence of magnesium ions, DNase I can randomly cut any site of double-stranded DNA, while in the presence of divalent manganese ions, DNase I can cut the DNA double strand at the same site to form a blunt end or a sticky end with 1 to 2 nucleotides protruding. In addition to double-stranded DNA hydrolysis activity, DNase I also has single-stranded DNA hydrolysis activity.

[0005] DNA enzymes are ubiquitous in the environment and in organisms, and have become a quality control issue of great concern to many molecular biology laboratories. The main reason is that the presence of DNA enzymes will degrade DNA molecules, which can easily cause degradation or contamination of experimental samples containing DNA, leading to the loss of experimental data, seriously affecting the experimental process and causing a waste of time and money. Therefore, DNA enzyme activity detection is a key quality control point for biological products, experimental reagents, and molecular biology laboratory environments.

[0006] There are currently a variety of methods for detecting the hydrolysis activity of DNase I. However, since DNase I can hydrolyze both single-stranded DNA and double-stranded DNA, the existing methods for detecting the hydrolysis activity of DNase I cannot distinguish the single-stranded DNA hydrolysis activity from the double-stranded DNA hydrolysis activity of DNase I. Therefore, there is an urgent need to find a method that can quantitatively detect the single-stranded DNA hydrolysis activity of DNase I with simple operation, accurate detection results and high sensitivity. Summary of the invention

[0007] In order to solve the problem that the existing DNA enzyme activity detection method is difficult to distinguish the single-stranded DNA hydrolysis activity and double-stranded DNA hydrolysis activity of DNA enzyme, and to improve the detection sensitivity and accuracy of DNA enzyme single-stranded DNA hydrolysis activity, the present invention provides a fluorescent probe for detecting DNA enzyme single-stranded DNA hydrolysis activity and its application.

[0008] According to a first aspect of the present invention, a fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzyme is provided. The fluorescent probe is a single-stranded DNA structure. The fluorescent probe contains a fluorescent group and a quencher group. The distance between the fluorescent group and the quencher group is 5 to 15 bp.

[0009] The present invention is based on the design of a universal fluorescent probe for a fluorescent RPA (recombinase polymerase amplification) system, and is based on single-stranded DNA. By simultaneously inserting a fluorescent group and a quenching group (single quenching group) into the single-stranded DNA and controlling the distance between the fluorescent group and the quenching group within the range of 5 to 15 bp, the fluorescent probe provided by the present invention is obtained through the above design, and the fluorescent probe is applied to the detection of DNA enzyme single-stranded hydrolysis activity. When the sample to be tested does not contain DNA enzyme or the DNA enzyme contained does not have single-stranded DNA hydrolysis activity, the structure of the fluorescent probe remains intact, the fluorescent group and the quenching group are close to each other, and no fluorescent signal is generated. When the sample to be tested contains DNA enzyme and the DNA enzyme has single-stranded DNA hydrolysis activity, the DNA enzyme with single-stranded DNA hydrolysis activity can hydrolyze the fluorescent probe, so that the fluorescent probe is broken by enzyme cleavage and the quenching group is dissociated. The unquenched fluorescent group emits a fluorescent signal due to its long distance from the quenching group. Therefore, the purpose of detecting the single-stranded hydrolysis activity of the DNA enzyme in the sample to be tested can be achieved by detecting the fluorescent signal in the detection system. Moreover, the fluorescent probe provided by the present invention has a single quenching group structure and there is a certain spatial distance between the quenching group and the fluorescent group, which is more in line with the single-stranded DNA hydrolysis characteristics in the DNA enzyme activity, and solves the problem that the fluorescent group and the quenching group are too far apart in the single-strand state, resulting in too high background noise during the detection process, and reduces the experimental error caused by incomplete hydrolysis to a certain extent, and improves the accuracy of the test results. In addition, the fluorescent probe provided by the present invention is specific to DNA enzymes with single-stranded DNA hydrolysis activity, and can specifically detect the single-stranded DNA enzyme hydrolysis activity, with high detection sensitivity, and the lowest detection limit of single-stranded DNA hydrolysis activity is as low as 0.0006 U / μL.

[0010] 5 bp is the shortest length that a DNA enzyme with single-stranded DNA hydrolysis activity can act on single-stranded DNA. If the distance between the fluorescent group and the quenching group in the fluorescent probe is less than 5 bp, the DNA enzyme with single-stranded DNA hydrolysis activity in the sample to be tested will not be able to act on the fluorescent probe, making it difficult for the quenching group in the fluorescent probe to dissociate from the fluorescent probe, and the structure of the fluorescent probe remains intact. The fluorescent group is quenched due to the close distance between the fluorescent group and the quenching group, and no fluorescent signal is generated, which ultimately makes the fluorescent probe unable to be used to effectively detect the single-stranded hydrolysis activity of the DNA enzyme, thereby reducing the accuracy of the detection of the single-stranded hydrolysis activity of the DNA enzyme; if the distance between the fluorescent group and the quenching group in the fluorescent probe is greater than 15 bp, the problem of too high detection background noise will occur in the process of using the fluorescent probe to detect the single-stranded hydrolysis activity of the DNA enzyme, thereby affecting the accuracy of the detection result.

[0011] Preferably, the length of the fluorescent probe is 12 to 18 bp.

[0012] By controlling the length of the fluorescent probe within the range of 12 to 18 bp, the background noise can be reduced in the process of detecting the single-strand hydrolysis activity of DNA enzyme using the fluorescent probe, thereby improving the accuracy of the detection results.

[0013] Preferably, the distance between the fluorescent group and the quencher group is 15 bp.

[0014] Preferably, the fluorescent group is located at the 5' end of the fluorescent probe, and the quencher group is located at the 3' end of the fluorescent probe, or, the fluorescent group is located at the 3' end of the fluorescent probe, and the quencher group is located at the 5' end of the fluorescent probe.

[0015] Preferably, the fluorescent group is located at the 5' end of the fluorescent probe, and the quencher group is located at the 3' end of the fluorescent probe.

[0016] Compared with inserting the fluorescent group and / or quenching group into the middle of the fluorescent probe, inserting the fluorescent group and / or quenching group into both ends of the fluorescent probe can reduce the synthesis cost of the fluorescent probe and thus reduce the detection cost of the single-stranded hydrolysis activity of the DNA enzyme.

[0017] Preferably, the fluorescent group is selected from one of FAM, ROX, JOE, Cy5 and VIC.

[0018] Preferably, the quenching group is selected from one of NFQ, BHQ1, BHQ2, and TAMRA.

[0019] Preferably, the fluorescent group is FAM and the quenching group is BHQ1.

[0020] Preferably, the nucleotide sequence of the fluorescent probe is as shown in SEQ ID NO:1.

[0021] According to the second aspect of the present invention, a method for detecting the single-chain hydrolysis activity of DNA enzyme is provided, comprising the following steps: incubating a sample to be tested with the above-mentioned fluorescent probe for detecting the single-chain hydrolysis activity of DNA enzyme, collecting the fluorescent signal, and judging the single-chain hydrolysis activity of DNA enzyme in the sample to be tested according to the intensity of the fluorescent signal.

[0022] The fluorescent probe provided by the present invention is applied to the detection of the single-chain hydrolysis activity of DNA enzyme. The purpose of detecting the single-chain hydrolysis of DNA enzyme can be achieved by detecting the intensity of the fluorescent signal in the detection system. The method for detecting the single-chain hydrolysis activity of DNA enzyme can detect a trace amount of DNA enzyme with single-chain DNA hydrolysis activity within 1 hour. The detection sensitivity is high, simple and fast, and the result is reliable. The method can simply, conveniently and quickly achieve the purpose of detecting the single-chain hydrolysis activity of DNA enzyme, and can also be used to detect the contamination level of DNA enzyme. The single-chain hydrolysis activity of DNA enzyme can be distinguished from the double-chain hydrolysis activity, which solves the problem that the single-chain DNA hydrolysis activity of DNA enzyme cannot be specifically determined in the prior art.

[0023] Preferably, the concentration of the fluorescent probe used to detect the single-stranded hydrolysis activity of DNA enzyme is 0.1-10 μmol / L.

[0024] Preferably, the incubation temperature is 25-45°C and the incubation time is 10-60 min.

[0025] Preferably, the method for collecting fluorescence signals includes fluorescence quantitative PCR instrument signal collection method, microwell microplate reader signal collection method, and spectrophotometer fluorescence signal collection method.

[0026] According to a third aspect of the present invention, a kit for detecting the single-strand hydrolysis activity of DNA enzyme is provided, and the kit comprises the above-mentioned fluorescent probe for detecting the single-strand hydrolysis activity of DNA enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a basic principle diagram for detecting the single-strand hydrolysis activity of DNA enzyme using the fluorescent probe provided by the present invention.

[0028] Figure 2 This is the original curve result diagram of the fluorescence intensity measurement of each group of samples at the 30th cycle using a real-time fluorescence PCR instrument in Test Example 1.

[0029] Figure 3 This is the original curve result diagram of the fluorescence intensity measurement of each group of samples at the 30th cycle using a real-time fluorescence PCR instrument in Test Example 2.

[0030] Figure 4This is the original curve result diagram of the fluorescence intensity measurement of the 30th cycle of each group of different concentrations of DNase I and double-stranded DNA enzyme using a real-time fluorescence PCR instrument in Test Example 3.

[0031] Figure 5 This is the original curve result diagram of the fluorescence intensity measurement of the 30th cycle of each group of different test samples using a real-time fluorescence PCR instrument in Test Example 4.

[0032] Figure 6 The standard curve diagram obtained by plotting for Test Example 4. DETAILED DESCRIPTION

[0033] The following is a further clear and complete description of the technical features in the technical solution provided by the present invention in conjunction with the specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0034] Example 1 Fluorescent probe for detecting DNA enzyme single-strand hydrolysis activity and its detection principle A fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzyme, the fluorescent probe is a single-stranded DNA structure with a length of 15 bp, and its nucleotide sequence is shown in SEQ ID NO: 1. The specific nucleotide sequence is as follows: 5′-CGCCATCGGAGGTTC-3′.

[0035] In this embodiment, a fluorescent group FAM is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 1, and a quenching group BHQ1 is inserted into the 3' end. The distance between the fluorescent group FAM and the quenching group is 15 bp. The specific nucleotide sequence of the fluorescent probe and the structure after the fluorescent group FAM and the quenching group BHQ1 are inserted are as follows: 5'-(FAM)-CGCCATCGGAGGTTC-(BHQ1)-3'.

[0036] The basic principle of using the fluorescent probe provided in this embodiment to detect the single-strand hydrolysis activity of DNA enzyme is as follows: Figure 1As shown, specifically as follows: when the sample to be tested does not contain DNA enzyme or the DNA enzyme contained does not have single-stranded DNA hydrolysis activity, the structure of the fluorescent probe remains intact, the fluorescent group and the quenching group are close to each other, and no fluorescent signal is generated; when the sample to be tested contains DNA enzyme and the DNA enzyme has single-stranded DNA hydrolysis activity, the DNA enzyme with single-stranded DNA hydrolysis activity can hydrolyze the fluorescent probe, so that the fluorescent probe is broken by enzyme cleavage and the quenching group is dissociated, and the unquenched fluorescent group emits a fluorescent signal due to its distance from the quenching group. Therefore, the purpose of detecting the single-stranded hydrolysis activity of the DNA enzyme in the sample to be tested can be achieved by detecting the fluorescent signal in the detection system.

[0037] Example 2 This embodiment provides a fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzymes. Compared with the embodiment 1, the difference in structure is that the length of the fluorescent probe is different.

[0038] The nucleotide sequence of the fluorescent probe provided in this embodiment is shown in SEQ ID NO: 2, with a length of 13 bp. The specific nucleotide sequence is as follows: 5′-CGCCATCGGAGGT-3′.

[0039] In this embodiment, a fluorescent group FAM is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 2, and a quenching group BHQ1 is inserted into the 3' end. The specific nucleotide sequence of the fluorescent probe and the structure after the fluorescent group FAM and the quenching group BHQ1 are inserted are as follows: 5'-(FAM)-CGCCATCGGAGGT-(BHQ1)-3'.

[0040] Except for the above differences, the structure and detection principle of the fluorescent probe provided in this embodiment are strictly consistent with those in Example 1.

[0041] Example 3 Method for detecting DNA enzyme single-strand hydrolysis activity A method for detecting DNA enzyme single-chain hydrolysis activity, comprising the following steps: incubating a sample to be tested with a fluorescent probe for detecting DNA enzyme single-chain hydrolysis activity at 25-45° C. for 10-60 min, collecting a fluorescent signal, and judging the DNA enzyme single-chain hydrolysis activity in the sample to be tested according to the intensity of the fluorescent signal; Among them, the concentration of the fluorescent probe used to detect the single-strand hydrolysis activity of DNA enzymes is 0.1~10 μmol / L; Methods for collecting fluorescence signals include fluorescence quantitative PCR instrument signal collection method, microwell enzyme marker signal collection method, and spectrophotometer fluorescence signal collection method.

[0042] Comparative Example 1 This comparative example provides a fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzymes. Compared with Example 1, the difference in structure is that the distance between the fluorescent group and the quenching group is different.

[0043] The nucleotide sequence of the fluorescent probe provided in this comparative example is shown in SEQ ID NO: 3, and the specific nucleotide sequence is as follows: 5'-ACAGCCTCAAGATCATCAGCAATGCCTCCT-3'.

[0044] In this comparative example, a fluorescent group FAM is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 3, and a quenching group BHQ1 is inserted into the 3' end. The distance between the fluorescent group FAM and the quenching group BHQ1 is 30 bp. The specific nucleotide sequence of the fluorescent probe and the structure after the insertion of the fluorescent group FAM and the quenching group BHQ1 are as follows: 5'-(FAM)-ACAGCCTCAAGATCATCAGCAATGCCTCCT-(BHQ1)-3'.

[0045] Except for the above differences, the structure and detection principle of the fluorescent probe provided in this comparative example are strictly consistent with those in Example 1.

[0046] Comparative Example 2 This comparative example provides a fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzymes. Compared with Example 1, the difference in structure is that the distance between the fluorescent group and the quenching group is different.

[0047] The nucleotide sequence of the fluorescent probe provided in this comparative example is shown in SEQ ID NO: 4, and the specific nucleotide sequence is as follows: 5'-TCATCTTCTAGTTGGTCTGTCTCCGCTTGG-3'.

[0048] In this comparative example, a fluorescent group FAM is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 4, and a quenching group BHQ1 is inserted into the 3' end. The distance between the fluorescent group FAM and the quenching group BHQ1 is 30 bp. The specific nucleotide sequence of the fluorescent probe and the structure after the insertion of the fluorescent group FAM and the quenching group BHQ1 are as follows: 5'-(FAM)-TCATCTTCTAGTTGGTCTGTCTCCGCTTGG-(BHQ1)-3'.

[0049] Except for the above differences, the structure and detection principle of the fluorescent probe provided in this comparative example are strictly consistent with those in Example 1.

[0050] Test Example 1 The effect of the length of the fluorescent probe and the distance between the fluorescent group and the quenching group on the detection results Table 1 Specific structures and sequences of different fluorescent probes

[0051] This test example aims to compare the detection effects of the fluorescent probes (as shown in Table 1) for detecting the single-stranded hydrolysis activity of DNA enzymes provided in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 in combination with the method for detecting the single-stranded hydrolysis activity of DNA enzymes provided in Example 3. The specific operation steps are as follows: 1. Preparation of Experimental Materials (1) Detection of DNA enzyme single-strand hydrolysis activity substrate The fluorescent probes for detecting the single-stranded hydrolysis activity of DNA enzymes provided by the synthesis of Example 1, Example 2, Comparative Example 1 and Comparative Example 2 are shown in Table 1.

[0052] The synthesized fluorescent probe was subjected to the following operation to obtain a detection substrate for DNA enzyme single-strand hydrolysis activity at a concentration of 0.1-10 μmol / L: A. Place the synthesized fluorescent probes ssDNA-1-FAM-BHQ1 (SEQ ID NO: 1), ssDNA-2-FAM-BHQ1 (SEQ ID NO: 2), ssDNA-1 (SEQ ID NO: 3), and ssDNA-2 (SEQ ID NO: 4) in brown centrifuge tubes, and centrifuge at 4°C and 8000 r / min for 2 min; B. Add 1×TE Buffer (Tris-EDTA buffer) to the brown centrifuge tubes containing ssDNA-1-FAM-BHQ1 (SEQ ID NO: 1), ssDNA-2-FAM-BHQ1 (SEQ ID NO: 2), ssDNA-1 (SEQ ID NO: 3), and ssDNA-2 (SEQ ID NO: 4) according to the label instructions to dilute the fluorescent probe concentration in each brown centrifuge tube to 100 μM; C. Use NonaDrop 8000 spectrophotometer to measure the concentrations of fluorescent probes ssDNA-1-FAM-BHQ1 (SEQ ID NO: 1), ssDNA-2-FAM-BHQ1 (SEQ ID NO: 2), ssDNA-1 (SEQ ID NO: 3), and ssDNA-2 (SEQ ID NO: 4); D. Continue to add 1×TE Buffer to the brown centrifuge tubes containing fluorescent probes ssDNA-1-FAM-BHQ1 (SEQ ID NO: 1), ssDNA-2-FAM-BHQ1 (SEQ ID NO: 2), ssDNA-1 (SEQ ID NO: 3), and ssDNA-2 (SEQ ID NO: 4), respectively, and adjust the concentration of the fluorescent probe to 0.1~10 µmol / L. Use the fluorescent probe at this concentration as a substrate for detecting the single-stranded hydrolysis activity of DNA enzymes in the test samples to detect the single-stranded hydrolysis activity of DNA enzymes.

[0053] (2) 10× Reaction Buffer Prepare 10× Reaction Buffer with a pH of 7.6 at 25°C according to the following composition: 100 mM Tris-HCl, 25 mM MgCl2, 5 mM CaCl2.

[0054] (3) DNA enzyme (DNase I) DNase I standard was purchased from Thermo Fisher Scientific Inc. (GIBCO TM , Invitrogen Corporation), catalog number: 18047019, and DNase I was used as a positive control.

[0055] (4) Enzyme-free water DNase / RNase-free distilled water (enzyme-free water) was purchased from UltraPure TM ), catalog number: 10977023, enzyme-free water was used as negative control.

[0056] 2. Experimental Instruments The fluorescence intensity was detected using AGS400 real-time fluorescence PCR instrument.

[0057] 3. Detection of DNA enzyme single-strand hydrolysis activity (1) Equilibrate 10× Reaction Buffer, 10× DNA enzyme single-strand hydrolysis activity detection substrate (fluorescent probe) and enzyme-free water to room temperature and mix them separately for use; DNase I (10 U / μL) should be placed in an ice bath for use and should be stored at 4°C immediately after use.

[0058] (2) Dilute 10× Reaction Buffer with enzyme-free water to 1× Reaction Buffer. Specifically, take 100 μL of 10× Reaction Buffer, add 900 μL of enzyme-free water, and mix well to obtain 1 mL of 1× Reaction Buffer.

[0059] (3) DNase I (10 U / μL) was diluted to an appropriate concentration gradient using 1× Reaction Buffer. The concentration gradient of DNase I was set to 0.0125 and 0 U / μL, respectively.

[0060] (4) Add each component and sample in sequence according to Table 2.

[0061] Table 2 Components and dosages in different fluorescent probe detection systems in Test Example 1

[0062] (5) Set the real-time fluorescence quantitative PCR reaction program according to the following conditions: 37°C, 60 s (signal collection) as one cycle, and run 30 cycles in total.

[0063] (6) The original curve was determined using a real-time fluorescence PCR instrument. The fluorescence intensity value of each group of samples at the 30th cycle was taken as the result. The fluorescence intensity detection results of each group of samples are shown in the following table. Figure 2 And as shown in Table 3.

[0064] Table 3 Fluorescence intensity of each gradient DNase I standard in test example 1 at the 30th cycle

[0065] Depend on Figure 2As shown in Table 3, when DNase I with a concentration of 0 U / μL is detected, the fluorescence intensity detected by the fluorescent probe ssDNA-1-FAM-BHQ1 provided in Example 1 is the lowest, and in the detection results of each group, when the fluorescent probe ssDNA-1-FAM-BHQ1 provided in Example 1 is used to detect DNase I with a concentration of 0 U / μL and DNase I with a concentration of 0.0125 U / μL, the fluorescence intensities of the two are greatly different and the difference between the fluorescence intensity values ​​of the two is the largest, when the fluorescent probe ssDNA-2-FAM-BHQ1 provided in Example 2 is used to detect DNase I with a concentration of 0 U / μL and DNase I with a concentration of 0.0125 U / μL, the fluorescence intensities of the two are also greatly different, but the difference between the fluorescence intensities of the two is lower than that of Example 1, and the fluorescent probe ssDNA-1 provided in Comparative Example 1 or the fluorescent probe ssDNA-2 provided in Comparative Example 2 respectively detects DNase I with a concentration of 0 U / μL and DNase I with a concentration of 0.0125 U / μL. When the fluorescence intensities of the two samples were detected with DNase I at a concentration of 0.0125 U / μL, they were close and the difference between the fluorescence intensity values ​​was extremely small. The main reason for the above results is that the distance between the fluorescent group FAM and the quenching group BHQ1 in the fluorescent probes ssDNA-1-FAM-BHQ1 and ssDNA-2-FAM-BHQ1 provided in Example 1 and Example 2, respectively, is between 5 and 15 bp, and the background noise in the detection process is low, which is suitable for being used as a substrate for detecting the single-stranded hydrolysis activity of DNA enzyme to detect the single-stranded hydrolysis activity of DNA enzyme, and the detection result is accurate; while the distance between the fluorescent group and the quenching group in the fluorescent probes provided in Comparative Example 1 and Comparative Example 2 is too far, both of which are 30 bp. In the process of detecting the single-stranded hydrolysis activity of DNA enzyme using the fluorescent probes provided in Comparative Example 1 and Comparative Example 2, the problem of too high detection background noise will occur, and therefore it is not suitable as a substrate for detecting the single-stranded hydrolysis activity of DNA enzyme to detect the single-stranded hydrolysis activity of DNA enzyme; the length of the fluorescent probe provided in Example 1 is 15 bp, and the length of the fluorescent probe provided in Example 2 is 13 bp, when the fluorescent probe provided in Example 2 is used to detect the single-stranded hydrolysis activity of DNA enzyme, the background noise is slightly higher than that in Example 1, and the accuracy of the detection result is slightly lower than that in Example 1, indicating that the background noise when the fluorescent probe with a length of 15 bp is used to detect the single-stranded hydrolysis activity of DNA enzyme is the lowest, and the accuracy of the detection result is the highest.

[0066] Test Example 2: Detection sensitivity test of fluorescent probe for detecting single-stranded DNA enzyme hydrolysis activity This test example aims to use the fluorescent probe for detecting the single-chain hydrolysis activity of DNA enzyme provided in Example 1 and the method for detecting the single-chain hydrolysis activity of DNA enzyme provided in Example 3 to detect different concentrations of DNA enzyme to determine the minimum detection limit, that is, the detection sensitivity, when detecting DNA enzyme and its single-chain hydrolysis activity using the fluorescent probe provided in Example 1.

[0067] Table 4 Components and dosages in the test system of Test Example 2 Components Dosage (volume) 1×Reaction Buffer 18 μL Samples to be tested (DNase I) 1 μL DNA enzyme single-strand hydrolysis activity detection substrate (fluorescent probe) 1 μL Total volume 20 μL Referring to the operating steps of Test Example 1, based on the upper and lower limits of the PCR instrument itself when detecting samples, DNase I (1 U / μL) was diluted with 1×Reaction Buffer to the following concentration gradient: 0.04, 0.02, 0.01, 0.005, 0.0025, 0.00125, 0.000625, 0.000013, 0 U / μL, and the detection system was prepared with reference to Table 4. The DNase I diluted to the above concentration gradient was detected using the fluorescent probe for detecting the single-stranded DNA enzyme detection activity provided in Example 1, wherein the real-time fluorescence quantitative PCR instrument reaction program was set as follows: 37° C., 60 s (signal collection) as 1 cycle, a total of 30 cycles were run, and the fluorescence intensity value of the 30th cycle of each group of samples was taken as the result. The fluorescence intensity detection results of the 30th cycle of DNase I of different concentrations in each group are as follows. Figure 3 And as shown in Table 5.

[0068] Table 5 Fluorescence intensity detection results of DNase I at different concentrations at the 30th cycle

[0069] Depend on Figure 3 As shown in Table 5, the fluorescent probe for detecting the single-strand hydrolysis activity of DNA enzyme provided in Example 1 can detect the single-strand hydrolysis activity of DNA enzyme in different concentration ranges, and has the characteristics of high sensitivity. The lowest detection limit of the single-strand hydrolysis activity of DNA enzyme is as low as 0.0006 U / μL.

[0070] Test Example 3 Specific detection of single-stranded DNA enzyme hydrolysis activity using fluorescent probes This test example aims to determine whether the fluorescent probe for detecting the single-strand hydrolysis activity of DNA enzyme provided in Probe Example 1 is specific for detecting DNA enzyme with single-strand hydrolysis activity.

[0071] Table 6 Components and dosages in the test system of Test Example 3

[0072] Referring to the operating steps of Test Example 1, DNase I (1 U / μL) was diluted with 1×Reaction Buffer to the following concentrations: 0.0125, 0 U / μL. At the same time, double-stranded DNA enzyme (1 U / μL) was diluted with 1×Reaction Buffer to the following concentrations: 0.0125, 0 U / μL. The detection system was prepared with reference to Table 6, and the fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzyme provided in Example 1 was used to detect the DNase I and double-stranded DNA enzyme diluted to the above concentrations. The fluorescence intensity detection results of DNase I and double-stranded DNA enzyme at different concentrations in each group at the 30th cycle are as follows: Figure 4 And as shown in Table 7.

[0073] Table 7 Fluorescence intensity detection results of DNase I and double-stranded DNA enzyme

[0074] From Table 7 and Figure 4 It can be seen that when DNase I and double-stranded DNA enzyme are detected using the fluorescent probe provided in Example 1, the fluorescence intensity is the highest when the detection concentration of DNase I is 0.0125 U / μL, and the fluorescence intensity is lower when the detection concentration of double-stranded DNA enzyme is 0.0125 U / μL. In addition, when DNase I with a concentration of 0 U / μL and DNase I with a concentration of 0.0125 U / μL are detected, the difference between the fluorescence intensity values ​​of the two is large, which proves that the fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzyme (DNA enzyme single-stranded hydrolysis activity detection substrate) provided in Example 1 has obvious specificity for the single-stranded DNA hydrolysis activity of DNA enzyme.

[0075] Test Example 4 Detection of DNA enzyme single-strand hydrolysis activity in different samples to be tested This test example aims to detect the single-strand hydrolysis activity of DNA enzyme in different samples using the fluorescent probe for detecting the single-strand hydrolysis activity of DNA enzyme provided in Example 1.

[0076] Table 8 Components and dosages in the test system of Test Example 4

[0077] Referring to the operating steps of Test Example 1, a standard curve was constructed with DNase I standard (Thermo Cat No. EN0521), and the single-stranded hydrolysis activity of DNA enzymes in different samples to be tested was detected. Specifically, DNase I (1 U / μL) was diluted with 1×Reaction Buffer to the following concentration gradient: 0.005, 0.0025, 0.00125, 0.000625, 0 U / μL, and each component and sample was added in sequence with reference to Table 8 for the experiment. The original curve was determined using a real-time fluorescence PCR instrument, and the fluorescence intensity value of the 30th cycle of each group of samples was taken as the result. The fluorescence intensity detection results of each group of samples are shown in FIG. Figure 5 As shown in Table 9, at the same time, according to the fluorescence intensity values ​​in Table 9, with RFU value as the y-axis and DNase I single-chain hydrolysis activity (U / μL) as the x-axis, a standard curve was drawn and an equation was fitted. The results are as follows Figure 6 As shown, the final equation of the standard curve is as follows: y = 2000000x + 377.05, R 2 =0.9987.

[0078] Table 9 Fluorescence intensity at the 30th cycle during the detection of each concentration gradient standard

[0079] Next, the single-strand hydrolysis activity of DNA enzyme in different samples to be tested was detected using the fluorescent probe for detecting the single-strand hydrolysis activity of DNA enzyme provided in Example 1, in combination with the method for detecting the single-strand hydrolysis activity of DNA enzyme provided in Example 3. The specific operation steps are as follows: Using the fluorescent probe provided in Example 1, the samples to be tested (RT reverse transcriptase, nuclease-free water, pure water, taq enzyme, and nuclease-free EP tube immersion solution) were detected according to the method provided in Example 3. The measurement results of the fluorescence intensity RFU value are shown in Table 10. The RFU value (y-axis coordinate) is substituted into the standard curve equation y = 2000000x + 377.05 obtained in this example, and the calculated x value (U / μL) is the DNase I content (single-strand hydrolysis activity of DNA enzyme) in the sample to be tested.

[0080] Table 10 DNA enzyme single-strand hydrolysis activity test results in different samples

[0081] As shown in Table 10, the RFU value (y-axis coordinate) is substituted into the standard curve equation y = 2000000x +377.05, and it is calculated that no single-chain hydrolysis activity of DNA enzyme was detected in nuclease-free water, pure water, and nuclease-free EP tube soaking solution. Specifically, the single-chain hydrolysis activity of DNA enzyme in nuclease-free water, pure water, and nuclease-free EP tube soaking solution is much less than the minimum detection limit of 0.0006 U / μL of the single-chain hydrolysis activity of DNA enzyme provided in Example 1; the concentration is 1mg / mL RT reverse transcriptase containing 0.0008 U / μL DNA enzyme single-chain hydrolysis activity, and the concentration is 1mg / mL taq enzyme containing 0.0009 U / μL DNA enzyme single-chain hydrolysis activity, wherein pure water and enzyme-free nucleic acid water require no DNase, which cannot be detected in theory and will be lower than the minimum detection limit. The above results show that the fluorescent probe provided by the present invention can be used to detect the single-chain hydrolysis activity of DNA enzyme in different samples to be tested.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting the single-strand hydrolysis activity of DNA enzyme, characterized in that: The fluorescent probe is a single-stranded DNA structure, and contains a fluorescent group and a quenching group, and the distance between the fluorescent group and the quenching group is 5-15 bp.

2. The fluorescent probe for detecting the single-stranded DNA enzyme hydrolysis activity according to claim 1, characterized in that: The length of the fluorescent probe is 12 to 18 bp.

3. The fluorescent probe for detecting the single-stranded DNA enzyme hydrolysis activity according to claim 2, characterized in that: The distance between the fluorescent group and the quenching group is 15 bp.

4. The fluorescent probe for detecting the single-stranded DNA enzyme hydrolysis activity according to claim 1, characterized in that: The fluorescent group is located at the 5' end of the fluorescent probe, and the quencher group is located at the 3' end of the fluorescent probe, or the fluorescent group is located at the 3' end of the fluorescent probe, and the quencher group is located at the 5' end of the fluorescent probe.

5. The fluorescent probe for detecting the single-stranded DNA enzyme hydrolysis activity according to claim 4, characterized in that: The fluorescent group is located at the 5' end of the fluorescent probe, and the quenching group is located at the 3' end of the fluorescent probe.

6. The fluorescent probe for detecting the single-stranded DNA enzyme hydrolysis activity according to claim 3, characterized in that: The nucleotide sequence of the fluorescent probe is shown in SEQ ID NO:

1.

7. The fluorescent probe for detecting the single-stranded DNA enzyme hydrolysis activity according to claim 1, characterized in that: The fluorescent group is FAM, and the quenching group is BHQ1.

8. A method for detecting the single-strand hydrolysis activity of DNA enzyme, characterized in that: The following steps are involved: The sample to be tested is incubated with the fluorescent probe for detecting the single-stranded hydrolysis activity of DNA enzyme as described in any one of claims 1 to 7, the fluorescent signal is collected, and the single-stranded hydrolysis activity of DNA enzyme in the sample to be tested is determined according to the intensity of the fluorescent signal.

9. The method for detecting the single-strand hydrolysis activity of DNA enzyme according to claim 8, characterized in that: The concentration of the fluorescent probe used to detect the single-stranded hydrolysis activity of DNA enzyme is 0.1-10 μmol / L; The incubation temperature is 25-45°C and the incubation time is 10-60 min.

10. A kit for detecting single-strand hydrolysis activity of DNA enzyme, characterized in that: The kit comprises a fluorescent probe for detecting single-stranded DNA enzyme hydrolysis activity as described in any one of claims 1 to 7.

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