Method for detecting relative activity of single-stranded DNA binding protein

Abstract

The invention provides a method for detecting the relative activity of a single-stranded DNA binding protein. The method comprises the following steps: incubating a to-be-detected sample and a fluorescent probe for detecting the single-stranded DNA binding protein, collecting a fluorescence signal, and judging the relative activity of the single-stranded DNA binding protein in the to-be-detected sample according to the intensity of the fluorescence signal, the fluorescent probe is of a single-stranded DNA structure, the length is 13-21 bp, and the fluorescent probe contains a reporter group and a quenching group; the reporter group and the quenching group are respectively positioned at the 5'end and the 3 'end of the fluorescent probe, or the reporter group and the quenching group are respectively positioned at the 3' end and the 5 'end of the fluorescent probe. The fluorescence intensity of the detection system after incubation of the to-be-detected sample and the fluorescent probe is detected so as to judge the relative activity of the single-stranded DNA protein in the to-be-detected sample, the purpose of detecting the relative activity of the single-stranded DNA binding protein can be achieved, the detection result is accurate, and the detection time is short.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for detecting the relative activity of a single-stranded DNA binding protein. Background Art

[0002] Single strand DNA binding protein (SSB or SSBP), also known as single strand binding protein, is a protein specifically responsible for binding to single strand regions of DNA. It is an essential component for DNA replication, recombination and repair. It can effectively keep the melted DNA duplexes separated (to prevent reannealing) and keep the two strands in single strand form. The polymerase chain reaction (PCR) can be optimized by adding SSB, which can stabilize denatured DNA, prevent duplex formation and protect single strand DNA (ssDNA) from digestion by nucleases. SSB can also be used for reverse transcription PCR and isothermal amplification of nucleic acids. In order to better apply SSB in molecular biology, it is often necessary to quantitatively detect its activity.

[0003] At present, the methods for detecting the activity of single-stranded DNA binding proteins are relatively limited. The commonly used method is electrophoretic mobility shift assay (EMSA), which mainly uses single-stranded DNA probes and SSBs for mixed incubation. The migration speed of the nucleic acid-protein complex in the sample in the polyacrylamide gel is slower than that of the free probe and forms a hysteresis band. The presence or absence of the hysteresis band on the gel is used to determine the presence or absence of single-stranded DNA binding proteins. This method can only be used for qualitative analysis and cannot be used for quantitative analysis of SSBs. Therefore, it is urgent to find a method that can quantitatively detect single-stranded DNA binding proteins and their relative activities. Summary of the invention

[0004] In order to solve the problem that the existing detection methods are difficult to quantitatively detect single-stranded DNA binding proteins and their relative activities, the present 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 a single-stranded DNA binding protein is provided, comprising the following steps: incubating a sample to be tested with a fluorescent probe for detecting the single-stranded DNA binding protein, collecting a fluorescent signal, and judging the relative activity of the single-stranded DNA binding protein in the sample to be tested according to the intensity of the fluorescent signal; the fluorescent probe is a single-stranded DNA structure, the length of the fluorescent probe is 13 to 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 a single-stranded DNA protein provided by the present invention determines the relative activity of the single-stranded DNA protein in the sample to be tested according to the fluorescence intensity in the detection system after the sample to be tested is incubated with a fluorescent probe for detecting the single-stranded DNA binding protein, wherein the fluorescent probe for detecting the single-stranded DNA binding protein is designed as a single-stranded DNA structure with a length within the range of 13 to 21 bp, and a reporter group is inserted into the 5' end and a quencher group is inserted into the 3' end, or a reporter group is inserted into the 3' end and a quencher group is inserted into the 5' end, and the fluorescent probe obtained by the above design is used for the detection of the relative activity of the single-stranded DNA binding protein. When the sample to be tested does not contain the single-stranded DNA binding protein, the sample to be tested cannot bind to the fluorescent probe, the structure of the fluorescent probe remains intact, and the reporter group and the quencher group are at a suitable distance (13 to 21 bp), the quenching group can quench the reporter group very well, and no fluorescent signal is generated in the detection system. When the sample to be tested contains single-stranded DNA binding protein, the single-stranded DNA binding protein in the sample to be tested will bind to the fluorescent probe, which will produce a blocking effect to hinder the quenching group from playing a quenching role, resulting in a reduction in the fluorescence quenching effect of the quenching group on the reporter group, and the reporter group is difficult to quench, thereby increasing the fluorescence intensity of the reporter group. The reporter group emits fluorescence and is detected. Therefore, by detecting the fluorescent signal in the detection system, the purpose of detecting the single-stranded DNA binding protein and its relative activity can be achieved, and it has the advantages of accurate detection results and short detection time.

[0007] The method for detecting the relative activity of a single-stranded DNA binding protein provided by the present invention can achieve the purpose of detecting the relative activity of a single-stranded DNA binding protein by controlling the length of the fluorescent probe used in the detection process to be between 13 and 21 bp, and the detection background noise is low when the relative activity of the single-stranded DNA binding protein is detected, which can improve the accuracy of the detection result. If the length of the fluorescent probe is too short (<13 bp), even if the sample to be tested contains single-stranded DNA binding protein, due to the short length of the fluorescent probe, the distance between the quenching group and the reporter group is too close, and the blocking effect produced when the single-stranded DNA binding protein binds to the above fluorescent probe has little effect on the quenching effect of the quenching group. The quenching group will directly quench the reporter group, resulting in a false negative in the test result; if the length of the fluorescent probe is too long (>21 bp), the distance between the quenching group and the reporter group is too far. Even if the sample to be tested does not contain single-stranded DNA binding protein, it is easy for the quenching group to fail to quench the reporter group. The reporter group will emit fluorescence, resulting in a false positive in the test result or insufficient linearity and discrimination of the test. The longer the length of the fluorescent probe, the greater the distance between the reporter group and the quenching group at both ends, the smaller the quenching effect of the quenching group on the reporter group, and the higher the background fluorescence, until there is no quenching effect at all, and the effect of detecting the relative activity of the single-stranded DNA binding protein cannot be achieved.

[0008] Preferably, the length of the fluorescent probe is 15 to 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°C.

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

[0014] Preferably, the content of the 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 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 This is a graph showing the result of testing the fluorescence signal intensity of the reaction system using the detection method provided in Example 1 (the nucleotide sequence is a fluorescent probe as shown in SEQ ID NO: 1) in Test Example 1.

[0020] Figure 3 This is a curve result diagram between the SSB content and the fluorescence intensity when the reaction system is detected using the detection method provided in Example 1 (the nucleotide sequence is a fluorescent probe shown in SEQ ID NO: 1) in Test Example 1.

[0021] Figure 4 This is a result diagram of test example 2 using the detection method provided in Example 2 (the nucleotide sequence is a fluorescent probe as shown in SEQ ID NO: 2) to detect the fluorescence signal intensity of the reaction system.

[0022] Figure 5 This is a result diagram of the fluorescent signal intensity of the reaction system detected by Test Example 2 using the detection method provided in Comparative Example 1 (the nucleotide sequence is a fluorescent probe as shown in SEQ ID NO: 3).

[0023] Figure 6 This is a result diagram of the fluorescent signal intensity of the reaction system detected by Test Example 2 using the detection method provided in Comparative Example 2 (the nucleotide sequence is a fluorescent probe shown in SEQ ID NO: 4).

[0024] Figure 7 This is a curve result diagram between the SSB content and the fluorescence intensity when the reaction system is detected using the detection method provided in Example 2 (the nucleotide sequence is a fluorescent probe shown in SEQ ID NO: 2) in Test Example 2.

[0025] Figure 8 This is a curve result diagram between SSB content and fluorescence intensity when the reaction system is detected using the detection method provided in Comparative Example 1 in Test Example 2 (the nucleotide sequence is a fluorescent probe as shown in SEQ ID NO: 3).

[0026] Fig. 9This is a curve result diagram between the SSB content and the fluorescence intensity when the reaction system is detected using the detection method provided in Comparative Example 2 (the nucleotide sequence is a fluorescent probe shown in SEQ ID NO: 4) in Test Example 2. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 A method for detecting the relative activity of a single-stranded DNA binding protein comprises the following steps: incubating a sample to be tested with a fluorescent probe for detecting the single-stranded DNA binding protein at 35-40° C. for 150-600 s, collecting a fluorescent signal, and judging the relative activity of the single-stranded DNA binding protein in the sample to be tested according to the intensity of the fluorescent signal, wherein the concentration of the fluorescent probe is 2.5-10 μM; The fluorescent probe used to detect single-stranded DNA binding protein 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: 5′-CGCCATCGGAGGTTC-3′.

[0029] By inserting a reporter group (ROX) at the 5' end and a quencher group (BHQ2) at the 3' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 1, the specific nucleotide sequence of the fluorescent probe and the structure after the reporter group ROX and the quencher group BHQ2 are inserted are as follows: 5'-ROX-CGCCATCGGAGGTTC-BHQ2-3'.

[0030] The method for detecting the relative activity of single-stranded DNA binding protein provided in this embodiment uses the above-mentioned fluorescent probe to detect the basic principle of single-stranded DNA binding protein. Figure 1As shown, specifically as follows: when the sample to be tested does not contain single-stranded DNA binding protein (SSB protein), the sample to be tested cannot bind to the fluorescent probe, the structure of the fluorescent probe remains intact, the distance between the reporter group and the quencher group meets 13~21 bp, the quencher group BHQ2 can have a good quenching effect on the reporter group ROX, and no fluorescent 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 to be tested will bind to the fluorescent probe, and a blocking effect will be generated to hinder the quenching effect of the quencher group, resulting in a decrease in the fluorescence quenching effect of the quencher group on the reporter group, and the reporter group is difficult to be quenched, thereby increasing the fluorescence intensity of the reporter group, and the reporter group emits fluorescence and is detected. Therefore, by detecting the fluorescent signal in the detection system, the purpose of detecting the single-stranded DNA binding protein and its relative activity can be achieved.

[0031] Example 2 This embodiment provides a method for detecting the relative activity of single-stranded DNA protein. Compared with Embodiment 1, the difference in structure is that the length and sequence of the fluorescent probe used to detect the single-stranded DNA binding protein in the detection process are different.

[0032] The length of the fluorescent probe used in this example is 20 bp, and its nucleotide sequence is shown in SEQ ID NO: 2. The specific nucleotide sequence is as follows: 5′-ACGATGCCGACCGGCGATGC-3′.

[0033] In this embodiment, a reporter group (ROX) is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 2, and a quencher group (BHQ2) is inserted into the 3' end. The specific nucleotide sequence of the fluorescent probe and the structure after the reporter group ROX and the quencher group BHQ2 are inserted are as follows: 5'-ROX-ACGATGCCGACCGGCGATGC-BHQ2-3' Except for the above differences, the method for detecting the relative activity of the single-stranded DNA binding protein provided in this embodiment, the structure of the fluorescent probe used, the detection principle, etc. are strictly consistent with those in Example 1.

[0034] Comparative Example 1 This comparative example provides a method for detecting the relative activity of a single-stranded DNA binding protein. Compared with Example 1, the difference in structure is that the fluorescent probe used to detect the single-stranded DNA binding protein in the detection process has a different length and sequence.

[0035] The length of the fluorescent probe used in this comparative example is 23 bp, and its nucleotide sequence is shown in SEQ ID NO: 3. The specific nucleotide sequence is as follows: 5'-CGCCATCTACTTGCCGAGCCAGG-3'.

[0036] In this comparative example, a reporter group (ROX) is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 3, and a quencher group (BHQ2) is inserted into the 3' end. The specific nucleotide sequence of the fluorescent probe and the structure after the reporter group ROX and the quencher group BHQ2 are inserted are as follows: 5'-ROX-CGCCATCTACTTGCCGAGCCAGG-BHQ2-3'.

[0037] Except for the above differences, the method for detecting the relative activity of the single-stranded DNA binding protein provided in this comparative example, the structure of the fluorescent probe used and the detection principle are strictly consistent with those in Example 1.

[0038] Comparative Example 2 This comparative example provides a method for detecting the relative activity of a single-stranded DNA binding protein. Compared with Example 1, the difference in structure is that the fluorescent probe used to detect the single-stranded DNA binding protein in the detection process has a different length and sequence.

[0039] The length of the fluorescent probe used in this comparative example is 25 bp, and its nucleotide sequence is shown in SEQ ID NO: 4. The specific nucleotide sequence is as follows: 5'-CAGCCCGTCCCGCCGATCTCGTCCA-3'.

[0040] In this comparative example, a reporter group (ROX) is inserted into the 5' end of the single-stranded DNA with a nucleotide sequence such as SEQ ID NO: 4, and a quencher group (BHQ2) is inserted into the 3' end. The specific nucleotide sequence of the fluorescent probe and the structure after the reporter group ROX and the quencher group BHQ2 are inserted are as follows: 5'-ROX-CAGCCCGTCCCGCCGATCTCGTCCA-BHQ2-3'.

[0041] Except for the above differences, the method for detecting the relative activity of single-stranded DNA binding protein provided in this comparative example, the structure of the fluorescent probe used and the detection principle are strictly consistent with those in Example 1. Test Example 1 This test example aims to detect the single-stranded DNA binding protein (SSB) and its relative activity in the sample to be tested using the method for detecting the relative activity of the single-stranded DNA binding protein provided in Example 1. The specific operation steps are as follows: 1. At room temperature, refer to Table 1 to add each component to the PCR tube to prepare a reaction system, wherein SSB is T4Gp32 single-stranded binding protein, purchased from Guangdong Guosheng Medical Technology Co., Ltd., with the catalog number GSP9601, and the test group is the test sample / test sample.

[0042] Table 1 Components and amounts of the reaction system of Test Example 1 using the detection method of Example 1

[0043] 2. After adding the above components, cover the PCR tube cap and vortex the PCR tube for 5-10 seconds to mix the components thoroughly. After centrifugation, add each group of PCR tubes to the PCR instrument.

[0044] 3. Select the emission wavelength channel corresponding to 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 shown.

[0045] Table 2 PCR instrument parameters during the detection process of Test Example 1

[0046] 4. Record the fluorescence intensity value of the 9th cycle of each group as the fluorescence intensity value of the group. The results are shown in Table 3.

[0047] Table 3 Detection results of fluorescence intensity values ​​of each group when testing Example 1 using the detection method of Example 1

[0048] From Table 3 and Figure 2 It can be seen that when the relative activity of the single-stranded DNA binding protein is detected using the method provided in Example 1, SSB can bind to the fluorescent probe whose nucleotide sequence is used therein, such as that shown in SEQ ID NO: 1, thereby hindering the quenching effect of the quenching group on the reporter group and increasing the fluorescence intensity of the reporter group. Furthermore, by detecting the fluorescence intensity in the detection system, the purpose of detecting the relative activity of SSB in the sample to be tested can be achieved.

[0049] 5. According to the test results in Table 3, a curve of SSB content and fluorescence intensity was drawn when 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 result deteriorated when the total amount of SSB exceeded 50 μg, 0-30 μg was taken as the linear interval of the standard curve, and the amount of SSB (μg) was taken as the abscissa (X-axis), and the fluorescence intensity value was taken as the ordinate (Y-axis). The following curve was drawn: Figure 3 The standard curve shown in the figure, the final linear formula is as follows: y = 101.73x + 58.722, R 2 = 0.9985. It can be seen from the standard curve and its linear formula that when making the standard curve, the content of the standard single-stranded DNA binding protein is between 0 and 30 μg. The obtained standard curve has a good linear relationship, which can improve the accuracy of detecting the samples using the standard curve.

[0050] The linear correlation R obtained in this embodiment 2 =0.998, with an excellent linear relationship, indicating that the method for detecting the relative activity of single-stranded DNA binding protein provided in Example 1 can well quantitatively detect the relative activity of single-stranded DNA binding protein due to the use of a fluorescent probe with a length of 15 bp, a reporter group of ROX, and a quencher group of BHQ2.

[0051] According to the above SSB activity standard curve y = 101.73x + 58.722, substituting the sample fluorescence intensity value, it can be calculated that the amount of SSB protein in the sample to be tested is 11.9 μg.

[0052] At present, there is no unified standard for defining the unit of SSB activity at home and abroad. In this test example, a reference method is provided. 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.

[0053] Test Example 2 This test example aims to detect the single-stranded DNA binding protein (SSB) and its relative activity in the sample to be tested using the method for detecting the relative activity of single-stranded DNA protein provided in Example 2, Comparative Example 1, and Comparative Example 2. The specific operation steps are as follows: 1. At room temperature, refer to Table 4, Table 5, and Table 6 to add various components to the PCR tube to prepare the reaction system, wherein SSB is T4Gp32 single-stranded binding protein, purchased from Guangdong Guosheng Medical Technology Co., Ltd., with the catalog number GSP9601.

[0054] Table 4 Components and amounts of the reaction system when testing in Test Example 2 using the detection method of Example 2

[0055] Table 5 Components and amounts of the reaction system when testing in Test Example 2 using the detection method of Comparative Example 1

[0056] Table 6 Components and amounts of the reaction system when testing in Test Example 2 using the detection method of Comparative Example 2

[0057] 2. After adding the above components, cover the PCR tube cap and vortex the PCR tube for 5-10 seconds to mix the components thoroughly. After centrifugation, add each group of PCR tubes to the PCR instrument.

[0058] 3. Select the emission wavelength channel corresponding to 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 shown.

[0059] Table 7 PCR instrument parameters in each detection process of Test Example 2

[0060] 4. Record the absolute fluorescence intensity value of the 9th cycle of each group (the value after the fluorescence intensity value stabilizes) as the fluorescence intensity value of the group. The results are shown in Table 8. Figure 4 , Figure 5 , Figure 6 As shown, the fluorescence signal intensity detection result 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) is as shown Figure 4 As shown, the fluorescence signal intensity detection result 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) is as shown in Figure 5 As shown, the fluorescence signal intensity detection result 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) is as follows Figure 6 shown.

[0061] Table 8 Fluorescence intensity test results of each group in Test Example 2

[0062] According to Table 8, Figure 4 , Figure 5 , Figure 6 As a result, curves of SSB content and fluorescence intensity were drawn when the detection method provided in Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 2), the detection method provided in Comparative Example 1 (the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 3), and the detection method provided in Comparative Example 2 (the nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 4). The results are shown respectively as follows: Figure 7 , Figure 8 , Fig. 9 shown.

[0063] By comparison Figure 3 and Figure 7 It can be seen that when SSB is detected using the detection method provided in Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 2), a good linear relationship is present when the SSB content is 0-10 μg, but the plateau phase will be entered prematurely. When the SSB content is above 10 μg, the linear relationship between the SSB protein content and the fluorescence intensity is poor. When SSB is detected using the detection method provided in Example 1 using a fluorescent probe with a nucleotide sequence such as SEQ ID NO: 1, a good linear relationship is present when the SSB content is 0-30 μg, and the method has the advantages of a wide detection range, low background noise, and good linearity. Therefore, the detection method provided in Example 1 is better than Example 2 in detecting SSB using a fluorescent probe with a nucleotide sequence such as SEQ ID NO: 1.

[0064] By comparison Figure 3 and Figure 8 Compared with Example 1, the detection method provided in Comparative Example 1 has a higher background noise when detecting SSB using a fluorescent probe with a nucleotide sequence such as SEQ ID NO: 3, and the linear relationship is not obvious, so it is not suitable for detecting the activity of SSB.

[0065] By comparison Figure 3 and Fig. 9 Compared with Example 1, when the detection method provided in Comparative Example 2 uses a fluorescent probe with a nucleotide sequence such as 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, and it is not suitable for detecting the activity of SSB.

[0066] In summary, the detection method provided in Example 1 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 1) and the detection method provided in Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 2) can both detect the relative activity of SSB, and the effect of detecting the relative activity of SSB using the detection method provided in Example 1 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 1) is better than the detection method provided in Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 2), while the detection method provided in Comparative Example 1 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 3) and the detection method provided in Comparative Example 2 (the nucleotide sequence of the fluorescent probe used is shown in SEQ ID NO: 4) are not suitable for detecting the relative activity of SSB.

[0067] 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 method for detecting the relative activity of a single-stranded DNA binding protein, characterized in that: The following steps are involved: Incubating the sample to be tested with a fluorescent probe for detecting single-stranded DNA binding protein, collecting fluorescent signals, and judging the relative activity of the single-stranded DNA binding protein in the sample to be tested according to the intensity of the fluorescent signals; 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.

2. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: The length of the fluorescent probe is 15-20 bp.

3. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 2, characterized in that: The nucleotide sequence of the fluorescent probe is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: 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.

5. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: The concentration of the fluorescent probe is 2.5-10 μM.

6. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: The temperature of the incubation operation is 35-40°C.

7. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: The incubation time is 150-600 s.

8. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: The content of the single-stranded DNA binding protein in the sample to be tested is 0-30 μg.

9. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 1, characterized in that: 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.

10. The method for detecting the relative activity of a single-stranded DNA binding protein according to claim 9, characterized in that: The reporter group is ROX, and the quencher group is BHQ2.

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