A method for detecting the relative activity of single-stranded DNA-binding proteins

Abstract

This invention provides a method for detecting the relative activity of single-stranded DNA-binding proteins. The method includes the following steps: incubating a test sample with a fluorescent probe for detecting single-stranded DNA-binding proteins, collecting fluorescence signals, and determining the relative activity of the single-stranded DNA-binding proteins in the test sample based on the intensity of the fluorescence signals; the fluorescent probe is a single-stranded DNA structure with a length of 13-21 bp, containing a reporter group and a quencher group; the reporter group and the quencher group are located at the 5' end and 3' end of the fluorescent probe, respectively, or the reporter group and the quencher group are located at the 3' end and 5' end of the fluorescent probe, respectively. This invention uses the fluorescence intensity of the detection system after incubation of the test sample with the fluorescent probe to determine the relative activity of the single-stranded DNA protein in the test sample, achieving the purpose of detecting the relative activity of single-stranded DNA-binding proteins, with accurate results and short detection time.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and specifically to a method for detecting the relative activity of single-stranded DNA-binding proteins. Background Technology

[0002] Single-stranded DNA-binding proteins (SSBs or SSBPs), also known as single-stranded binding proteins, are proteins specifically responsible for binding to single-stranded regions of DNA. They are essential components for DNA replication, recombination, and repair, effectively maintaining the separation of unwinding DNA duplexes (preventing re-annealing) and keeping both strands in single-stranded form. Polymerase chain reaction (PCR) can be optimized by adding SSBs. SSBs stabilize denatured DNA, prevent duplex formation, and protect single-stranded DNA (ssDNA) from digestion by nucleases. SSBs can also be used for reverse transcription PCR and isothermal amplification of nucleic acids. To better utilize SSBs in molecular biology, quantitative detection of their activity is often necessary.

[0003] Currently, methods for detecting the activity of single-stranded DNA-binding proteins (SSBs) are relatively limited. A commonly used method is electrophoretic mobility shift assay (EMSA), which involves incubating a mixture of single-stranded DNA probes and SSBs. The nucleic acid-protein complexes in the sample migrate more slowly on the polyacrylamide gel than the free probes, forming a hysteresis band. The presence or absence of this hysteresis band indicates the presence of SSBs. However, this method is only suitable for qualitative analysis and cannot quantify SSBs. Therefore, there is an urgent need to find a method for the quantitative detection of single-stranded DNA-binding proteins and their relative activity. Summary of the Invention

[0004] To address the problem that existing detection methods are insufficient for the quantitative detection of single-stranded DNA-binding proteins and their relative activity, this invention provides a method for detecting the relative activity of single-stranded DNA-binding proteins.

[0005] According to a first aspect of the present invention, a method for detecting the relative activity of single-stranded DNA-binding proteins is provided, comprising the following steps: incubating a test sample with a fluorescent probe for detecting single-stranded DNA-binding proteins, collecting a fluorescence signal, and determining the relative activity of the single-stranded DNA-binding proteins in the test sample based on the intensity of the fluorescence signal; wherein the fluorescent probe is a single-stranded DNA structure, the length of the fluorescent probe is 13-21 bp, and the fluorescent probe contains a reporter group and a quencher group; the reporter 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 reporter 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.

[0006] The method for detecting the relative activity of single-stranded DNA proteins provided by this invention determines the relative activity of single-stranded DNA proteins in the test sample by detecting the fluorescence intensity in the detection system after incubation of the test sample with a fluorescent probe used to detect single-stranded DNA binding proteins. The fluorescent probe for detecting single-stranded DNA binding proteins is designed as a single-stranded DNA structure with a length in the range of 13-21 bp, and has a reporter group inserted at the 5' end and a quencher group inserted at the 3' end, or vice versa. When the fluorescent probe designed as described above is used to detect the relative activity of single-stranded DNA binding proteins, if the test sample does not contain single-stranded DNA binding proteins, the test sample cannot bind to the fluorescent probe, the structure of the fluorescent probe remains intact, and the reporter group and quencher group are at a suitable distance (13-21 bp). Within a bp range, the quencher group effectively quenches the reporter group, resulting in no fluorescence signal in the detection system. When the sample contains single-stranded DNA-binding proteins, these proteins bind to the fluorescent probe, creating a blocking effect that hinders the quencher group from exerting its quenching effect. This reduces the fluorescence quenching effect of the quencher group on the reporter group, making it difficult to quench the reporter group. Consequently, the fluorescence intensity of the reporter group increases, leading to its fluorescence emission and detection. Therefore, detecting the fluorescence signal in the detection system allows for the detection of single-stranded DNA-binding proteins and their relative activity, offering advantages such as accurate results and short detection time.

[0007] The method for detecting the relative activity of single-stranded DNA binding proteins provided by this invention achieves the purpose of detecting the relative activity of single-stranded DNA binding proteins by controlling the length of the fluorescent probe used in the detection process between 13 and 21 bp. Furthermore, the detection noise is low when detecting the relative activity of single-stranded DNA binding proteins, which can improve the accuracy of the detection results. If the length of the fluorescent probe is too short (<13 bp), even if the sample contains single-stranded DNA binding proteins, the quencher group and reporter group will be too close due to the short probe length. The blocking effect of the single-stranded DNA binding protein when it binds to the fluorescent probe will have little impact on the quenching effect of the quencher group, and the quencher group will directly quench the reporter group, leading to false negatives. If the length of the fluorescent probe is too long (>21 bp), the quencher group and reporter group will be too far apart. Even if the sample does not contain single-stranded DNA binding proteins, the quencher group may not be able to quench the reporter group, and the reporter group will emit fluorescence, leading to false positives or insufficient linearity and discrimination. Furthermore, the longer the fluorescent probe, the greater the distance between the reporter group and the quencher group at both ends, the smaller the quenching effect of the quencher group on the reporter group, and the higher the background fluorescence, until there is no quenching effect at all, failing to achieve the effect of detecting the relative activity of single-stranded DNA binding proteins.

[0008] Preferably, the length of the fluorescent probe is 15~20 bp.

[0009] Preferably, the nucleotide sequence of the fluorescent probe is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] Preferably, the reporter 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.

[0011] Preferably, the concentration of the fluorescent probe is 2.5~10 μM.

[0012] Preferably, the incubation temperature is 35~40℃.

[0013] Preferably, the incubation time is 150~600 s.

[0014] Preferably, the content of single-stranded DNA-binding protein in the sample to be tested is 0~30 μg.

[0015] Preferably, the reporter group is selected from one of FAM, ROX, CY5, and VIC, and the quencher group is selected from one of BHQ1 and BHQ2.

[0016] Preferably, the reporter group is ROX and the quencher group is BHQ2.

[0017] Preferably, the methods for acquiring fluorescence signals include the fluorescence quantitative PCR instrument signal acquisition method, the microplate reader signal acquisition method, and the spectrophotometer fluorescence signal acquisition method. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the principle of relative activity detection for single-stranded DNA-binding proteins.

[0019] Figure 2 The image shows the results of detecting the fluorescence signal intensity of the reaction system using the detection method provided in Example 1 (a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO: 1).

[0020] Figure 3 The graph shows the curve results of SSB content versus fluorescence intensity when the reaction system was detected using the detection method provided in Example 1 (fluorescent probe with nucleotide sequence as shown in SEQ ID NO: 1).

[0021] Figure 4 The result of detecting the fluorescence signal intensity of the reaction system using the detection method provided in Example 2 (fluorescent probe with nucleotide sequence as shown in SEQ ID NO: 2) is shown in Figure 2.

[0022] Figure 5 The image shows the results of detecting the fluorescence signal intensity of the reaction system using the detection method provided in Comparative Example 1 (a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO: 3) in Test Example 2.

[0023] Figure 6 The result of detecting the fluorescence signal intensity of the reaction system using the detection method provided in Comparative Example 2 (fluorescent probe with nucleotide sequence as shown in SEQ ID NO: 4) is shown in Figure 2.

[0024] Figure 7 The graph shows the curve results of SSB content versus fluorescence intensity when the reaction system was detected using the detection method provided in Example 2 (fluorescent probe with nucleotide sequence as shown in SEQ ID NO: 2).

[0025] Figure 8 The graph shows the curve results of SSB content versus fluorescence intensity when the reaction system was detected using the detection method provided in Comparative Example 1 (fluorescent probe with nucleotide sequence as shown in SEQ ID NO: 3) in Test Example 2.

[0026] Figure 9The graph shows the curve results of SSB content versus fluorescence intensity when the reaction system was detected using the detection method provided in Comparative Example 2 (fluorescent probe with nucleotide sequence as shown in SEQ ID NO: 4). Detailed Implementation

[0027] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1

[0029] A method for detecting the relative activity of single-stranded DNA binding proteins includes the following steps: incubating the sample to be tested with a fluorescent probe for detecting single-stranded DNA binding proteins at 35-40°C for 150-600 s, collecting fluorescence signals, and determining the relative activity of single-stranded DNA binding proteins in the sample to be tested based on the intensity of the fluorescence signals, wherein the concentration of the fluorescent probe is 2.5-10 μM.

[0030] The fluorescent probe used above for detecting single-stranded DNA-binding proteins is a single-stranded DNA structure with a length of 15 bp. Its nucleotide sequence is shown in SEQ ID NO: 1. The specific nucleotide sequence is as follows:

[0031] 5'-CGCCATCGGAGGTTC-3'.

[0032] By inserting a reporter group (ROX) at the 5' end and a quencher group (BHQ2) at the 3' end of the single-stranded DNA nucleotide sequence as shown in SEQ ID NO: 1, the specific nucleotide sequence of the fluorescent probe and the structure after the insertion of the reporter group ROX and the quencher group BHQ2 are as follows:

[0033] 5'-ROX-CGCCATCGGAGGTTC-BHQ2-3'.

[0034] The method for detecting the relative activity of single-stranded DNA-binding proteins provided in this embodiment utilizes the basic principle of detecting single-stranded DNA-binding proteins using the aforementioned fluorescent probes, as follows: Figure 1As shown, specifically: When the sample to be tested does not contain single-stranded DNA-binding protein (SSB protein), the sample cannot bind to the fluorescent probe, the structure of the fluorescent probe remains intact, the distance between the reporter group and the quencher group is 13~21 bp, the quencher group BHQ2 can effectively quench the reporter group ROX, and no fluorescence signal is generated in the detection system; When the sample to be tested contains single-stranded DNA-binding protein (SSB protein), the single-stranded DNA-binding protein (SSB protein) in the sample will bind to the fluorescent probe, producing a blocking effect that hinders the quencher group from exerting its quenching effect, resulting in a reduced fluorescence quenching effect of the quencher group on the reporter group, making it difficult for the reporter group to be quenched, thereby increasing the fluorescence intensity of the reporter group, and the reporter group emits fluorescence and is thus detected. Therefore, by detecting the fluorescence signal in the detection system, the purpose of detecting single-stranded DNA-binding protein and its relative activity can be achieved.

[0035] Example 2

[0036] This embodiment provides a method for detecting the relative activity of single-stranded DNA proteins. Compared with Example 1, the difference lies in the length and sequence of the fluorescent probe used to detect single-stranded DNA binding proteins during the detection process.

[0037] The fluorescent probe used in this embodiment is 20 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 2. The specific nucleotide sequence is as follows:

[0038] 5'-ACGATGCCGACCGGCGATGC-3'.

[0039] In this embodiment, a reporter group (ROX) is inserted at the 5' end and a quencher group (BHQ2) is inserted at the 3' end of the single-stranded DNA nucleotide sequence shown in SEQ ID NO: 2. The specific nucleotide sequence of the fluorescent probe and the structure after the insertion of the reporter group ROX and the quencher group BHQ2 are as follows:

[0040] 5'-ROX-ACGATGCCGACCGGCGATGC-BHQ2-3'

[0041] Apart from the differences mentioned above, the method for detecting the relative activity of single-stranded DNA binding proteins, the structure of the fluorescent probe used, and the detection principle provided in this embodiment are all strictly consistent with those in Example 1.

[0042] Comparative Example 1

[0043] This comparative example provides a method for detecting the relative activity of single-stranded DNA binding proteins. The difference between this method and Example 1 is that the fluorescent probes used to detect single-stranded DNA binding proteins have different lengths and sequences.

[0044] The fluorescent probe used in this comparative example is 23 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 3. The specific nucleotide sequence is as follows:

[0045] 5'-CGCCATCTACTTGCCGAGCCAGG-3'.

[0046] This comparative example involves inserting a reporter group (ROX) at the 5' end and a quencher group (BHQ2) at the 3' end of a single-stranded DNA nucleotide sequence as shown in SEQ ID NO: 3. The specific nucleotide sequence of the fluorescent probe and the structure after the insertion of the reporter group ROX and the quencher group BHQ2 are as follows:

[0047] 5'-ROX-CGCCATCTACTTGCCGAGCCAGG-BHQ2-3'.

[0048] Apart from the differences mentioned above, the method for detecting the relative activity of single-stranded DNA binding proteins, the structure of the fluorescent probes used, and the detection principle provided in this comparative example are all strictly consistent with those in Example 1.

[0049] Comparative Example 2

[0050] This comparative example provides a method for detecting the relative activity of single-stranded DNA binding proteins. The difference between this method and Example 1 is that the fluorescent probes used to detect single-stranded DNA binding proteins have different lengths and sequences.

[0051] The fluorescent probe used in this comparative example is 25 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 4. The specific nucleotide sequence is as follows:

[0052] 5'-CAGCCCGTCCCGCCGATCTCGTCCA-3'.

[0053] This comparative example involves inserting a reporter group (ROX) at the 5' end and a quencher group (BHQ2) at the 3' end of a single-stranded DNA nucleotide sequence as shown in SEQ ID NO: 4. The specific nucleotide sequence of the fluorescent probe and the structure after the insertion of the reporter group ROX and the quencher group BHQ2 are as follows:

[0054] 5'-ROX-CAGCCCGTCCCGCCGATCTCGTCCA-BHQ2-3'.

[0055] Apart from the differences mentioned above, the method for detecting the relative activity of single-stranded DNA-binding proteins, the structure of the fluorescent probe used, and the detection principle provided in this comparative example are all strictly consistent with those in Example 1.

[0056] Test Example 1

[0057] This test example aims to detect the single-stranded DNA binding protein (SSB) and its relative activity in the test sample using the method for detecting the relative activity of single-stranded DNA binding proteins provided in Example 1. The specific operating steps are as follows:

[0058] 1. Under room temperature conditions, add each component to the PCR tube according to Table 1 to prepare the reaction system. SSB is T4Gp32 single-chain binding protein, purchased from Guangdong Guosheng Medical Technology Co., Ltd., catalog number GSP9601. The test group is the test sample / sample to be tested.

[0059] Table 1. Components and their amounts in the reaction system using the detection method of Example 1 in Test Example 1.

[0060]

[0061] 2. After adding the above components, cap the PCR tubes and vortex the PCR tubes for 5-10 seconds until all components are fully mixed. After centrifugation, add each group of PCR tubes into the PCR instrument.

[0062] 3. Select the channel corresponding to the emission wavelength of the reporter group contained in each fluorescent probe, and detect the fluorescence signal intensity of the reaction system according to the parameters shown in Table 2. The results are as follows: Figure 2 As shown.

[0063] Table 2 PCR instrument parameters during the detection process of Test Example 1

[0064]

[0065] 4. Record the fluorescence intensity value of each group in the 9th cycle as the fluorescence intensity value of that group. The results are shown in Table 3.

[0066] Table 3. Detection results of fluorescence intensity values ​​for each group when using the detection method of Example 1 in Test Example 1.

[0067]

[0068] From Table 3 and Figure 2 It is known that when the relative activity of single-stranded DNA binding protein is detected using the method provided in Example 1, SSB can bind to the fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 1, which prevents the quenching group from quenching the reporter group and increases the fluorescence intensity of the reporter group. Thus, by detecting the fluorescence intensity in the detection system, the relative activity of SSB in the test sample can be detected.

[0069] 5. Based on the test results in Table 3, plot the curve of SSB content versus fluorescence intensity during detection using the detection method of Example 1 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 1). Because the linearity of the detection results deteriorates when the total amount of SSB exceeds 50 μg, 0~30 μg is taken as the linear range of the standard curve. The amount of SSB (μg) is used as the abscissa (X-axis), and the fluorescence intensity value is used as the ordinate (Y-axis), as shown in the figure. Figure 3 The standard curve shown yields the following linear formula: y = 101.73x + 58.722, R0 2 = 0.9985. As can be seen from the standard curve and its linear formula, when the content of single-stranded DNA binding protein in the standard is between 0 and 30 μg, the obtained standard curve has a good linear relationship, which can improve the accuracy of the test sample detection using the standard curve.

[0070] The linear correlation R obtained in this embodiment 2 =0.998, indicating excellent linearity. This demonstrates that the method provided in Example 1 for detecting the relative activity of single-stranded DNA binding proteins can effectively quantify the relative activity of single-stranded DNA binding proteins by using a fluorescent probe with a length of 15 bp, a reporter group of ROX, and a quencher group of BHQ2.

[0071] Based on the above SSB activity standard curve y = 101.73x + 58.722, and substituting the sample fluorescence intensity value, the amount of SSB protein in the sample to be tested can be calculated to be 11.9 μg.

[0072] Currently, there is no unified standard for defining SSB activity units, either domestically or internationally. This test example provides a reference method, and the empirical formula is as follows: the relative activity at a standard SSB content of 10 μg is 100 U, that is, the SSB protein activity (U) in the test sample = SSB protein amount ÷ (10 / 100). Therefore, substituting into the empirical formula, the SSB protein activity in the test sample with an SSB protein amount of 11.9 μg is 119 U, and the SSB protein activity unit in the test sample is 39.66 U / μL.

[0073] Test Example 2

[0074] This test example aims to detect the relative activity of single-stranded DNA proteins in the test sample and its relative activity using the methods provided in Example 2, Comparative Example 1, and Comparative Example 2. The specific operating steps are as follows:

[0075] 1. Under room temperature conditions, add each component to the PCR tube according to Tables 4, 5 and 6 to prepare the reaction system. SSB is T4Gp32 single-chain binding protein, purchased from Guangdong Guosheng Medical Technology Co., Ltd., catalog number GSP9601.

[0076] Table 4. Components and their amounts in the reaction system when tested using the detection method of Example 2.

[0077]

[0078] Table 5. Components and their amounts in the reaction system when tested using the detection method of Comparative Example 1 in Test Example 2.

[0079]

[0080] Table 6. Components and their amounts in the reaction system when tested using the detection method of Comparative Example 2 in Test Example 2.

[0081]

[0082] 2. After adding the above components, cap the PCR tubes and vortex the PCR tubes for 5-10 seconds until all components are fully mixed. After centrifugation, add each group of PCR tubes into the PCR instrument.

[0083] 3. Select the channel corresponding to the emission wavelength of the reporter group contained in each fluorescent probe, and detect the fluorescence signal intensity of the reaction system according to the parameters shown in Table 7. The results are as follows: Figure 4 , Figure 5 , Figure 6 As shown.

[0084] Table 7 PCR instrument parameters for each detection process in Test Example 2

[0085]

[0086] 4. Record the absolute fluorescence intensity value (the value after the fluorescence intensity value has stabilized) of each group in the 9th cycle, and use it as the fluorescence intensity value of that group. The results are shown in Table 8. Figure 4 , Figure 5 , Figure 6 As shown, the fluorescence signal intensity detection results of the reaction system using the detection method of Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 2) are as follows. Figure 4 As shown, the fluorescence signal intensity detection results of the reaction system using the detection method of Comparative Example 1 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 3) are as follows: Figure 5As shown, the fluorescence signal intensity detection results of the reaction system using the detection method of Comparative Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 4) are as follows: Figure 6 As shown.

[0087] Table 8. Detection results of fluorescence intensity values ​​for each group in Test Example 2

[0088]

[0089] According to Table 8, Figure 4 , Figure 5 , Figure 6 As a result, curves showing the SSB content versus fluorescence intensity were plotted for the detection methods provided in Example 2 (the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 2), Comparative Example 1 (the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 3), and Comparative Example 2 (the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 4). The results are shown in the figures below. Figure 7 , Figure 8 , Figure 9 As shown.

[0090] By comparison Figure 3 and Figure 7 It can be seen that when using the detection method provided in Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 2) to detect SSB, there is a good linear relationship when the SSB content is 0~10 μg, but it will enter the plateau phase too early. When the SSB content is above 10 μg, the linear relationship between SSB protein content and fluorescence intensity is poor. However, when using the detection method provided in Example 1 to detect SSB with the fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 1, there is a good linear relationship between the SSB content and 30 μg, which has the advantages of wide detection range, low background noise and good linearity. Therefore, the detection method provided in Example 1 is better than that of Example 2 when using the fluorescent probe with the nucleotide sequence shown in SEQ ID NO: 1 to detect SSB.

[0091] By comparison Figure 3 and Figure 8 Compared to Example 1, the detection method provided in Comparative Example 1 has a higher noise floor and no obvious linearity when using a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO: 3 to detect SSB, and is not suitable for detecting the activity of SSB.

[0092] By comparison Figure 3 and Figure 9Compared to Example 1, the detection method provided in Comparative Example 2, when using a fluorescent probe with a nucleotide sequence as shown in SEQ ID NO: 4 to detect SSB, although there is a certain linear relationship between the SSB content and the fluorescence intensity, the linearity is poor and the data fluctuates greatly, making it unsuitable for detecting the activity of SSB.

[0093] In summary, both the detection methods provided in Example 1 (with the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 1) and Example 2 (with the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 2) can detect the relative activity of SSB. The detection method provided in Example 1 (with the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 1) is superior to the detection method provided in Example 2 (with the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 2). The detection methods provided in Comparative Example 1 (with the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 3) and Comparative Example 2 (with the nucleotide sequence of the fluorescent probe shown in SEQ ID NO: 4) are not suitable for detecting the relative activity of SSB.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A method for detecting the relative activity of single-stranded DNA-binding proteins, characterized in that, Includes the following steps: The sample to be tested is incubated with a fluorescent probe for detecting single-stranded DNA-binding proteins, and the fluorescence signal is collected. The relative activity of the single-stranded DNA-binding proteins in the sample to be tested is determined based on the intensity of the fluorescence signal. The fluorescent probe is a single-stranded DNA structure with a length of 15 bp, and the fluorescent probe is 5'-ROX-CGCCATCGGAGGTTC-BHQ2-3'.

2. The method for detecting the relative activity of single-stranded DNA-binding proteins as described in claim 1, characterized in that: The concentration of the fluorescent probe is 2.5~10 μM.

3. The method for detecting the relative activity of single-stranded DNA-binding proteins as described in claim 1, characterized in that: The incubation operation is performed at a temperature of 35~40℃.

4. The method for detecting the relative activity of single-stranded DNA-binding proteins as described in claim 1, characterized in that: The incubation period is 150-600 seconds.

5. The method for detecting the relative activity of single-stranded DNA-binding proteins as described in claim 1, characterized in that: The content of single-stranded DNA-binding protein in the sample to be tested is 0~30 μg.

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