Method and kit for detecting activity of recombinase

Abstract

The invention relates to the field of biological detection, in particular to a recombinase activity detection method and a kit. According to the method for detecting the strand displacement activity of the recombinase, the strand displacement template is subjected to fluorescence labeling, a quenching group modified DNA strand is displaced by utilizing the strand displacement effect of the recombinase, and the relative activity of the recombinase to be detected is obtained through a linear fitting equation of the fluorescence intensity increasing rate and the activity. The method can be used for detecting strand displacement products through a fluorescence detection means, is non-toxic and harmless, is simple and feasible, and can be used for determining the strand displacement recombination activity of the recombinase within 1 hour. By using the method, quantitative data of strand displacement activity can be obtained, and the strand displacement activity of recombinase can be accurately analyzed.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a detection method and a kit for recombinase activity. Background Art

[0002] Homologous recombination is very important for error-free repair of DNA double-strand breaks and restart of replication forks. Recombinases of the RecA / Rad51 family play a core catalytic role in this process. Bacteriophage recombinase (UvsX) is a type of recombinase protein that has been studied extensively. It is widely used and can be applied to isothermal amplification of recombinases, and can quickly achieve amplification from a single molecule of template to a detectable level. At present, the test method for the strand displacement activity of recombinases mainly adopts isotope labeling or agarose gel electrophoresis, and the recombinase activity is determined by analyzing the strand displacement products. However, this method is very harmful, has poor experimental reproducibility, and cannot be accurately quantified. In addition, the reaction substrate fragments used in this method are relatively long, and single-strand binding proteins need to be added. The system is relatively complex and the cost is relatively high.

[0003] Therefore, providing a method for testing the recombinase chain displacement activity has important practical significance. Summary of the invention

[0004] In view of this, the present invention provides a method and kit for detecting recombinase activity. The method fluorescently labels the primer sequence, utilizes the strand displacement effect of the recombinase to displace the nucleic acid chain modified with the quenching group, and obtains the relative activity of the recombinase to be tested by the linear fitting equation of the fluorescence intensity increase rate and the activity. The method can detect the product of strand displacement by means of fluorescence detection, is non-toxic and harmless, simple and easy to operate, and can determine the strand displacement recombination activity of the recombinase within 1 hour; and the inspection process uses a substrate with a fluorescent primer, without the need to add a single-stranded binding protein, simplifying the system components and reducing costs. The method can obtain quantitative data of strand displacement activity, and can accurately analyze the strand displacement activity of the recombinase.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for detecting recombinase activity, comprising the following steps:

[0007] (1) obtaining a strand displacement template, wherein the strand displacement template comprises a first nucleic acid molecule and a second nucleic acid molecule that is at least partially complementary to the first nucleic acid molecule, wherein a fluorescent group is disposed at the 5' end of the first nucleic acid molecule and a quenching group is disposed at the 3' end of the second nucleic acid molecule;

[0008] (2) mixing a recombinase standard, a strand displacement primer and the strand displacement template, and detecting the fluorescence value of the mixed system within a given period of time, wherein the strand displacement primer is complementary to at least a portion of the first nucleic acid molecule;

[0009] (3) establishing a linear fitting equation y=ax+b of the absolute value of the first fluorescence value change per unit time and the enzyme activity based on the change of the fluorescence value, wherein y is the absolute value of the fluorescence value change, x is the enzyme concentration or enzyme activity, and a and b are constants;

[0010] (4) Determine the activity of the recombinant enzyme to be tested based on the linear fitting equation.

[0011] In some specific embodiments of the present invention, the determining the activity of the recombinase to be tested comprises:

[0012] The recombinase to be tested is substituted for the recombinase standard, and the fluorescence value within a given time period is obtained according to the method of step (1) and step (2);

[0013] Based on the change in the fluorescence value, determining an absolute value of a change in a second fluorescence value per unit time;

[0014] Based on the absolute value of the change in the second fluorescence value and the linear fitting equation y=ax+b, the activity of the recombinase to be tested is determined.

[0015] In some specific embodiments of the present invention, when the recombinase to be tested includes two or more recombinases, determining the activity of the recombinase to be tested includes:

[0016] Respectively obtaining the absolute value of the change in the second fluorescence value of two or more recombinases to be tested with the same concentration within a unit time;

[0017] Comparing the absolute values ​​of the changes in the second fluorescence values ​​of two or more recombinases to be tested;

[0018] Based on the comparison results, the activity ranking of the two or more recombinases to be tested is determined.

[0019] In some specific embodiments of the present invention, the given time period is within 5-10 minutes after mixing the recombinase standard, the strand displacement primer and the strand displacement template.

[0020] In some specific embodiments of the present invention, the mixed recombinase, strand displacement primer and strand displacement template include:

[0021] mixing the recombinase and the strand displacement primer, and incubating to obtain a mixture;

[0022] The mixture is mixed with the strand displacement template.

[0023] In some specific embodiments of the present invention, the incubation time is 0-10 min.

[0024] In some specific embodiments of the present invention, the unit time is 20-40s; and / or

[0025] The given period of time is within 5-10 minutes after mixing the mixture with the strand displacement template.

[0026] In some specific embodiments of the present invention, the method for detecting the fluorescence value within a given time period is: detecting the fluorescence value once every 20-35 seconds, taking the previous N detection results, where N is an integer of 5-20.

[0027] In some specific embodiments of the present invention, the complementarity percentage between the first nucleic acid molecule and the second nucleic acid molecule is greater than or equal to 80%.

[0028] In some specific embodiments of the present invention, the percentage of complementarity between the first nucleic acid molecule and the second nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the first nucleic acid molecule and the second nucleic acid molecule are completely complementary.

[0029] In some specific embodiments of the present invention, the complementarity percentage between the strand displacement primer and the first nucleic acid molecule is greater than or equal to 80%.

[0030] In some specific embodiments of the invention, the strand displacement primer is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the first nucleic acid molecule, or the strand displacement primer is completely complementary to the first nucleic acid molecule.

[0031] In some specific embodiments of the present invention, the recombinase is selected from bacteriophage T4 Uvs X recombinase, bacteriophage RB69 Uvs X recombinase, bacteriophage Aeh1 Uvs X recombinase, bacteriophage KVP40 Uvs X recombinase, and Escherichia coli RecA recombinase.

[0032] In some specific embodiments of the present invention, the step of mixing the recombinase standard, the strand displacement primer and the strand displacement template comprises mixing the strand displacement primer and the strand displacement template at a molar concentration ratio of 1:1 to 1:5.

[0033] In some specific embodiments of the present invention, the mixed system further comprises: at least one of a buffer, magnesium ions, Tris-OAC or a crowding agent.

[0034] In some specific embodiments of the present invention, in the mixed system, the concentration of magnesium ions is 6-20 mmol / L.

[0035] In some specific embodiments of the present invention, the crowding agent is selected from at least one of PEG, PVP and dextran.

[0036] In some specific embodiments of the present invention, the average molecular weight of the PEG is in the range of 4000-10000;

[0037] The average molecular weight of the PVP is in the range of 10000-60000;

[0038] The average molecular weight of the dextran is in the range of 10,000-70,000.

[0039] In some specific embodiments of the present invention, the volume concentration of the crowding agent is 2%-5.5%.

[0040] In some specific embodiments of the present invention, the quenching group is BHQ, the fluorescent group is FAM, 484 nm is used as the excitation light, and 530 nm is used as the emission light to detect the fluorescence value within a given period of time.

[0041] In a second aspect, the present invention provides a kit for detecting recombinase activity, comprising:

[0042] A strand displacement template, the strand displacement template comprising a first nucleic acid molecule and a second nucleic acid molecule that is at least partially complementary to the first nucleic acid molecule, the 5' end of the first nucleic acid molecule is provided with a fluorescent group, and the 3' end of the second nucleic acid molecule is provided with a quenching group;

[0043] a strand displacement primer that is complementary to at least a portion of the first nucleic acid molecule; and

[0044] Recombinant enzyme standards.

[0045] In some specific embodiments of the present invention, the complementarity percentage between the first nucleic acid molecule and the second nucleic acid molecule is greater than or equal to 80%;

[0046] Optionally, the percent complementarity between the first nucleic acid molecule and the second nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the first nucleic acid molecule and the second nucleic acid molecule are completely complementary.

[0047] In some specific embodiments of the present invention, the complementarity percentage between the strand displacement primer and the first nucleic acid molecule is greater than or equal to 80%.

[0048] In some specific embodiments of the invention, the strand displacement primer is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the first nucleic acid molecule, or the strand displacement primer is completely complementary to the first nucleic acid molecule.

[0049] In some specific embodiments of the present invention, the recombinase is selected from bacteriophage T4 Uvs X recombinase, bacteriophage RB69 Uvs X recombinase, bacteriophage Aeh1 Uvs X recombinase, bacteriophage KVP40 Uvs X recombinase, and Escherichia coli RecA recombinase.

[0050] In some specific embodiments of the present invention, in the kit, the molar concentration ratio of the strand displacement primer to the strand displacement template is 1:1 to 1:5.

[0051] In some specific embodiments of the present invention, the kit further comprises: at least one of a buffer, magnesium ions, Tris-OAC or a crowding agent.

[0052] In some specific embodiments of the present invention, the concentration of the magnesium ions in the mixed system formed by the kit is 6-20 mmol / L.

[0053] In some specific embodiments of the present invention, the crowding agent is selected from at least one of PEG, PVP and dextran.

[0054] In some specific embodiments of the present invention, the average molecular weight of the PEG is in the range of 4000-10000;

[0055] The average molecular weight of the PVP is in the range of 10000-60000;

[0056] The average molecular weight of the dextran is in the range of 10,000-70,000.

[0057] In some specific embodiments of the present invention, the volume concentration of the crowding agent in the mixed system formed by the kit is 2%-5.5%.

[0058] In summary, the present invention provides a method for determining the chain displacement activity of a recombinase protein, which mainly includes: (1) preparing a chain displacement template; (2) incubating and combining a recombinase sample and a chain displacement primer; (3) preparing a chain displacement reaction system and detecting the reaction fluorescence; (4) data analysis and activity calculation. The method fluorescently labels the chain displacement template, utilizes the chain displacement effect of the recombinase to displace a nucleic acid chain modified with a quenching group, such as a DNA chain, and obtains the relative activity of the recombinase to be tested by a linear fitting equation of the fluorescence intensity increase rate and the activity. The method can determine the chain displacement recombination activity of the recombinase within 1 hour; and the inspection process uses a substrate with a fluorescent primer, without the need to add a single-stranded binding protein, thereby simplifying the system components and reducing costs. The method can obtain quantitative data on the chain displacement activity, and can accurately analyze the chain displacement activity of the recombinase.

[0059] The present invention also provides a detection substrate for recombinase activity, comprising a chain displacement template and a chain displacement primer, wherein one chain of the chain displacement template is FAM fluorescently modified, and the other complementary chain is BHQ quenching modified, and the BHQ quenching group quenches the FAM fluorescence, causing the chain displacement template to not fluoresce, and after adding the chain displacement primer, the chain with fluorescence modification can be displaced under the action of the recombinase, and then the change of FAM fluorescence value is detected by an enzyme marker, the increase of the fluorescence value is proportional to the chain displacement activity, and the activity of the unknown sample can be calculated using the change relationship of the fluorescence value of the standard.

[0060] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0061] (1) A substrate with a fluorescent primer was designed to detect the strand displacement product by fluorescence detection. This method is non-toxic, simple and easy to use, and can measure the strand displacement recombination activity of the recombinase within 1 hour.

[0062] (2) This method can be used to obtain quantitative data on strand displacement activity, and can accurately analyze the strand displacement activity of the recombinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0064] Figure 1 A graph showing the change in fluorescence values ​​of the recombinant enzyme UvsX of different concentrations of the standard provided in Example 5 of the present invention over time;

[0065] Figure 2 A standard curve diagram of the concentration-slope of the standard recombinant enzyme UvsX at three different time periods provided in Example 5 of the present invention;

[0066] Figure 3 A graph showing the change in fluorescence value of the standard recombinant enzyme UvsX over time at different magnesium ion concentrations provided in Example 6 of the present invention;

[0067] Figure 4 A bar graph showing the relationship between different magnesium ion concentrations and slopes at two different time periods provided in Example 6 of the present invention;

[0068] Figure 5 A graph showing the change in fluorescence value of the standard recombinant enzyme UvsX over time at different crowding reagent concentrations provided in Example 7 of the present invention;

[0069] Figure 6 A bar graph showing the relationship between the concentration of different crowding reagents and the slope at three different time periods provided in Example 7 of the present invention;

[0070] Figure 7 A graph showing the change in fluorescence value of the standard recombinase UvsX over time at different strand displacement template to strand displacement primer concentration ratios provided in Example 8 of the present invention;

[0071] Figure 8 A bar graph showing the relationship between the concentration ratios of different strand displacement templates and strand displacement primers and the slopes at five different time periods provided in Example 8 of the present invention;

[0072] Fig. 9 This is a standard curve diagram provided for Example 9 of the present invention. DETAILED DESCRIPTION

[0073] The present invention discloses a detection method and a kit for recombinase activity. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0074] Explanation of terms

[0075] "Recombinase" has single-stranded DNA binding activity. When there is a double-stranded DNA fragment with a target sequence that is homologously complementary to the single-stranded DNA molecule in the system, the recombinase with strand displacement activity can bind to the single-stranded DNA molecule to form a stable complex under the condition that ATP or ATP analogs provide energy. The complex seeks and invades the double-stranded DNA region that is homologously complementary to the single-stranded DNA molecule sequence, and a strand displacement reaction occurs under the action of the recombinase. In the present invention, since the single-stranded DNA molecule contains a sequence that is homologously complementary to a portion of the double-stranded DNA fragment, a partial strand displacement reaction occurs on the double-stranded DNA fragment.

[0076] "Fluorophore" or "fluorescent label" refers to any substance that emits electromagnetic energy of a certain wavelength (emission wavelength) when irradiated with radiation of a different wavelength (excitation wavelength), and is intended to cover chemical or biochemical molecules or fragments thereof that are capable of specifically interacting or reacting with an analyte of interest in a sample to provide one or more optical signals.

[0077] Representative fluorophores for use in the methods provided herein include, for example, FAM, (tetramethylrhodamine) Texas Red™, green fluorescent protein, blue fluorescent protein, red fluorescent protein, fluorescein, 5-fluorescein isothiocyanate (FITC), cyanine dyes (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Bodipy dyes (Invitrogen), and / or Alexa Fluor Fluor dyes (Invitrogen), dansyl, dansyl chloride (DNS-C1), 5-(iodoacetamide) fluorescein (5-IAF), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenz-2-oxadiazol-4-yl chloride (NBD-Cl), ethidium bromide, fluorescein, rhodamine dyes (5-carboxyrhodamine 6G hydrochloride, lissamine rhodamine B sulfonyl chloride, rhodamine-B-isothiocyanate (RITC), rhodamine 800); tetramethylrhodamine 5- (and 6-) isothiocyanate (TRITC)), Texas RedTM, sulfonyl chloride, naphthylaminesulfonic acid (including but not limited to 1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS)), anthracene fatty acids, DPH, octadecatetraenoic acid, TMA-DPH, fluorenyl fatty acids, fluorescein-phosphatidylethanolamine, Texas red-phosphatidylethanolamine, pyrenyl-phosphatidylcholine, fluorenyl-phosphatidylcholine, merocyanine 540, naphthylstyryl, 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-l-methylpyridinium salt (di-5-ASP), Cy-3 iodoacetamide, Cy-5-N-hydroxysuccinimide, Cy-7-isothiocyanate, IR-125, thiazole orange, azure B, Nile blue, aluminum phthalocyanine, oxazine 1,4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolate (MQAE), Fura-2, calcium green, carboxy SNARF-6, BAPTA, coumarin, phytofiuors, coronene, and metal-ligand complexes.

[0078] A "quencher group" or "quencher label" is also considered a detectable label.

[0079] The terms "first" and "second" are used only for descriptive purposes to distinguish purposes such as substances, components, etc. from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the embodiments of the present application, the first nucleic acid molecule may also be referred to as the second nucleic acid molecule, and correspondingly, the second nucleic acid molecule is referred to as the first imaging process. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0080] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0081] The present invention provides a method for detecting recombinase activity, comprising the following steps:

[0082] S1: obtaining a strand displacement template, the strand displacement template comprising a first nucleic acid molecule and a second nucleic acid molecule that is at least partially complementary to the first nucleic acid molecule, wherein a fluorescent group is disposed at the 5' end of the first nucleic acid molecule and a quenching group is disposed at the 3' end of the second nucleic acid molecule;

[0083] S2: mixing a recombinase standard, a strand displacement primer and a strand displacement template, and detecting the fluorescence value of the mixed system within a given period of time, wherein the strand displacement primer is complementary to at least a portion of the first nucleic acid molecule;

[0084] S3: Based on the change of fluorescence value, a linear fitting equation y=ax+b is established between the absolute value of the change of the first fluorescence value per unit time and the enzyme activity, wherein y is the absolute value of the change of fluorescence value, x is the enzyme concentration or enzyme activity, and a and b are constants;

[0085] S4: Based on the linear fitting equation, the activity of the recombinant enzyme to be tested is determined.

[0086] Specifically, the strand displacement template provided in step S1 is a reaction substrate of a recombinase, which includes two complementary nucleic acid molecule chains (a first nucleic acid molecule and a second nucleic acid molecule). The two nucleic acid molecule chains tend to have sequences of equal length, and the nucleotides at at least one end of the strand displacement template are complementary. In some embodiments, the nucleotides at both ends of the strand displacement template are complementary.

[0087] The 5' end of one nucleic acid molecule chain, such as the first nucleic acid molecule, is fluorescently modified, and the 3' end of the other nucleic acid molecule chain, such as the second nucleic acid molecule, is quenched, so that the strand displacement template is in a fluorescence inhibition state, that is, the strand displacement template does not emit fluorescence when excited. It should be understood that the first nucleic acid molecule and the second nucleic acid molecule can be interchangeable, such as the 3' end of the first nucleic acid molecule is fluorescently modified, and the 5' end of the second nucleic acid molecule is quenched. In the embodiment of the present invention, the terminal nucleotide of the first nucleic acid molecule that is fluorescently modified is complementary to the terminal nucleotide of the second nucleic acid molecule that is quenched, thereby fully exerting the quenching effect of the quenching modification on the fluorescence modification.

[0088] In the embodiment of the present invention, the quenching modification can be achieved by adding a quenching group, and the fluorescence modification can be achieved by adding a fluorescent group. The principle of the quenching group and the fluorescent group in the detection is: in the strand displacement template, since the quenching group and the fluorescent group are located at the same end of the template bimolecule, the quenching group can reduce or eliminate the fluorescence emission of the surrounding adjacent fluorescent groups, resulting in the strand displacement template not generating fluorescence. By adding a strand displacement primer to the reaction system, under the action of the recombinase, the nucleic acid molecule chain with the quenching modification can be displaced, so that the new nucleic acid molecule chain formed after the displacement has the fluorescent luminescence property again. Therefore, the embodiment of the present invention does not limit the selection of the quenching group and the fluorescent group. For example, the fluorescence modification can be modified by FAM, of course, it is not limited thereto; the quenching modification can be modified by BHQ, and the BHQ quenching group can quench the FAM fluorescence, so that the strand displacement template does not generate fluorescence. Of course, the type of the quenching group is not limited to BHQ.

[0089] The inventors of the present application found in experiments that in the strand displacement template, the lower the homology of the two nucleic acid molecule chains, the lower the efficiency of the strand displacement reaction by heating the recombinase. In one embodiment, the complementarity percentage between the first nucleic acid molecule and the second nucleic acid molecule is greater than or equal to 80%. The higher the homology, the more obvious the strand displacement effect. In some embodiments, the complementarity percentage between the first nucleic acid molecule and the second nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the first nucleic acid molecule and the second nucleic acid molecule are completely complementary. The complementarity percentage referred to in the embodiments of the present invention refers to the number of bases that are complementary through base pairing in two complementary double-stranded nucleic acid molecules, as a percentage of the total number of bases in the double-stranded nucleic acid molecule.

[0090] In the above step S2, the strand displacement primer is a nucleic acid molecule that is complementary to at least a portion of the first nucleic acid molecule. In one embodiment, the complementary percentage between the strand displacement primer and the first nucleic acid molecule is greater than or equal to 80%. In some embodiments, the complementary percentage between the strand displacement primer and the first nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the strand displacement primer and the first nucleic acid molecule are completely complementary. In some embodiments, the complementary percentage between the strand displacement primer and the first nucleic acid molecule is greater than the complementary percentage between the second nucleic acid molecule and the first nucleic acid molecule, so as to promote the strand displacement reaction and improve the strand displacement efficiency.

[0091] In the embodiment of the present invention, the recombinase standard refers to a recombinase sample with a known enzyme concentration or enzyme activity. When the recombinase standard is a recombinase sample with a known enzyme concentration, the enzyme concentration can be used as a parameter to collect data for subsequent analysis, or the enzyme concentration can be converted to enzyme activity according to the conversion relationship between enzyme concentration and enzyme activity in this step, and then the data is collected for subsequent analysis. The enzyme concentration referred to in the embodiment of the present invention can be the mass content of the recombinase in a unit volume, or it can be the molar content of the recombinase in a unit volume. In the embodiment of the present invention, 1 unit of enzyme activity refers to the amount of enzyme that can convert 1 micromole of substrate within 1 minute under specific conditions (such as 25° C.), or the amount of enzyme (U) that converts 1 micromole of related groups in the substrate.

[0092] The recombinase standard includes a plurality of samples with gradient enzyme concentrations or gradient enzyme activities, the number of samples is 3 or more, and in some embodiments, the recombinase standard may be provided with five or more samples with gradient enzyme concentrations or gradient enzyme activities. In one embodiment, the recombinase standard is formed by preparing a recombinase with known enzyme activity into a plurality of samples with different concentrations and known concentrations.

[0093] The recombinase standard, the strand displacement primer and the strand displacement template are mixed to form a mixed system. At this time, under the action of the recombinase, the strand displacement primer can displace the nucleic acid molecule chain with quenching modification in the strand displacement primer, such as the second nucleic acid molecule, and the strand displacement primer and the nucleic acid molecule chain with fluorescence modification form a new nucleic acid molecule double strand. Since the new nucleic acid molecule double strand does not contain a quenching group, the fluorescent group on the first nucleic acid molecule generates fluorescence.

[0094] In some embodiments, mixing a recombinase standard, a strand displacement primer and a strand displacement template includes: mixing a recombinase and a strand displacement primer, incubating to obtain a mixture; mixing the mixture with a strand displacement template. Firstly mixing the recombinase and the strand displacement primer to improve the uniformity of the two, and then adding the strand displacement template can improve the regional uniformity of the reaction, avoid some areas reacting rapidly due to too high a concentration of the recombinase and / or the strand displacement primer, and the reaction may be completed in a very short time; while some areas react slowly due to too low a concentration of the recombinase and / or the strand displacement primer. Both of the above situations are not conducive to obtaining regular changes in fluorescence values.

[0095] In some embodiments, the step of mixing the recombinase standard, the strand displacement primer and the strand displacement template is to mix the strand displacement primer and the strand displacement template at a molar concentration ratio of 1:1 to 1:5. The inventors of the present application found in experiments that when the molar concentration of the strand displacement primer is increased to an excess (up to 3 times the excess), the recombination displacement efficiency is improved. Exemplarily, the recombinase standard, the strand displacement primer and the strand displacement template are mixed at a molar concentration ratio of 1:1, 1:2, 1:3, 1:4, 1:5.

[0096] In some embodiments, at least one of a buffer, magnesium ions, Tris-OAC, and a crowding agent is mixed with a recombinase standard, a strand displacement primer, and a strand displacement template to provide favorable conditions for the strand displacement enzyme to exert its strand displacement activity. In some embodiments, the concentration of magnesium ions in the mixed system is 6-20 mmol / L, and exemplary, the concentration of magnesium ions can be 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, and the like.

[0097] In some embodiments, the crowding agent is a macromolecular polymer selected from at least one of PEG, PVP and dextran. In some embodiments, the average molecular weight range of PEG is 4000-10000, and PEG is selected from PEG4000, PEG6000, PEG8000, PEG10000, PEG20000, PEG35000, and PEG100000. In some embodiments, the average molecular weight range of PVP is 10000-60000, and PVP is selected from PVP10000, PVP24000, PVP40000, and PVP55000. In some embodiments, the average molecular weight range of dextran is 10000-70000, and dextran is selected from dextran 10000, dextran 20000, dextran 40000, and dextran 70000.

[0098] In some embodiments, the volume concentration of the crowding agent is 2%-5.5%. For example, the volume concentration of the crowding agent can be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, etc. The inventors of the present application also found in experiments that the recombination replacement efficiency can be improved by optimizing the concentration of the crowding agent and the Mg ion.

[0099] In some embodiments, the incubation time of the mixed recombinase and the strand displacement primer is 0-10 minutes. During the above incubation time, fluorescence collection can be performed to collect fluorescence intensity suitable for measurement and reflect the change of fluorescence intensity per unit time. Exemplary, the incubation time is 1s, 5s, 10s, 15s, 30s, 45s, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, etc.

[0100] The inventors found that in the mixed system, the fluorescence value increases with the enhancement of the recombinase chain displacement activity, and the two are positively correlated. Therefore, the activity of the unknown sample can be calculated using the fluorescence value change relationship of the standard. In view of this, the embodiment of the present invention detects the change in the fluorescence value of the fluorescent group through a fluorescence intensity detector such as an ELISA instrument, and records the fluorescence value within a given time period. In some embodiments, when the quenching group is BHQ and the fluorescent group is FAM, 484nm is used as the excitation light and 530nm is used as the emission light to detect the fluorescence value within a given time period.

[0101] In some embodiments, the given time period is within 5-10 minutes after the mixture and the strand displacement template are mixed. At this time, the displacement enzyme can reflect the relationship between the absolute value of the change in fluorescence value per unit time and the enzyme activity during the period of sufficient strand displacement reaction. Exemplarily, the given time period is 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, and 10 minutes after the mixture and the strand displacement template are mixed.

[0102] In some embodiments, the method for detecting the fluorescence value within a given time period is: the fluorescence value is detected once every 20-35 seconds, and the previous N detection results are taken to construct the absolute value of the first fluorescence value change per unit time. Wherein N is an integer of 5 to 20. It should be understood that as the given time period is extended, the number of sampling times can be increased.

[0103] In step S3, in an embodiment of the present application, the absolute value of the change in the first fluorescence value per unit time within a given time period is obtained. In some embodiments, the unit time is 20-40s. Exemplarily, the unit time can be 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s, 30s, 31s, 32s, 32s, 33s, 34s, 35s, 36s, 37s, 38s, 40s and the like.

[0104] The fitting equation y=ax+b is fitted based on the absolute value of the first fluorescence value change per unit time of different enzyme activities and the corresponding mixed system. Wherein, x is the enzyme concentration or enzyme activity, y is the absolute value of the fluorescence value change, when the test sample is a recombinant enzyme standard, y corresponds to the absolute value of the first fluorescence value change; when the test sample is the recombinant enzyme to be tested, y corresponds to the absolute value of the second fluorescence value change. When x is the enzyme concentration, y=ax+b is the fitting equation between the absolute value of the fluorescence value change per unit time of the recombinant enzyme and the enzyme concentration of the recombinant enzyme; when x is the enzyme activity, y=ax+b is the fitting equation between the absolute value of the fluorescence value change per unit time of the recombinant enzyme and the enzyme activity of the recombinant enzyme.

[0105] In step S4, the activity of the recombinase to be tested can be determined based on the linear fitting equation and the absolute value of the fluorescence value change of the mixed system containing the recombinase to be tested per unit time.

[0106] In some embodiments, the activity of the recombinase to be tested includes: replacing the recombinase standard with the recombinase to be tested, and obtaining the fluorescence value of the mixed system containing the recombinase to be tested within a given time period according to the method of step S1 and step S2; determining the absolute value of the change of the second fluorescence value per unit time based on the change of the fluorescence value; determining the activity of the recombinase to be tested based on the absolute value of the change of the second fluorescence value and the linear fitting equation y=ax+b. Exemplarily, when calculating the activity of the recombinase to be tested, y in the linear fitting equation y=ax+b is the absolute value of the change of the second fluorescence value, that is, the absolute value of the change of the fluorescence value of the mixed system containing the recombinase to be tested. Specifically, the absolute value of the change of the second fluorescence value is substituted into y in the linear fitting equation, and x in y=ax+b is calculated to obtain the activity of the recombinase to be tested.

[0107] In some embodiments, when x is the enzyme concentration, the method further includes converting the calculated concentration of the recombinant enzyme to be tested into enzyme activity according to the conversion relationship between enzyme concentration and enzyme activity.

[0108] In one embodiment, when the recombinase to be tested includes two or more recombinases, the activity strength of the two or more recombinases can be determined by the method provided in the embodiment of the present application. In one embodiment, when the recombinase to be tested includes two or more recombinases, determining the activity of the recombinase to be tested includes: obtaining the absolute value of the change in the second fluorescence value of each of the two or more recombinases to be tested of the same concentration per unit time; comparing the absolute value of the change in the second fluorescence value of the two or more recombinases to be tested; based on the comparison results, determining the activity ranking of the two or more recombinases to be tested.

[0109] In the embodiment of the present application, the recombinase to be tested and the recombinase in the recombinase standard are the same recombinase. Exemplarily, the recombinase is selected from bacteriophage T4 Uvs X recombinase, bacteriophage RB69 Uvs X recombinase, bacteriophage Aeh1 Uvs X recombinase, bacteriophage KVP40 Uvs X recombinase, Escherichia coli RecA recombinase, but is not limited thereto.

[0110] The embodiment of the present invention fluorescently labels the primer sequence, utilizes the chain displacement effect of the recombinase to displace the DNA chain modified with the quenching group, and obtains the relative activity of the recombinase to be tested through the linear fitting equation of the fluorescence intensity increase rate and the activity. The method can detect the product of chain displacement by fluorescence detection means, is non-toxic and harmless, simple and easy to operate, and can determine the chain displacement recombination activity of the recombinase within 1 hour; and the inspection process uses a substrate with a fluorescent primer, without the need to add a single-stranded binding protein, simplifying the system components and reducing costs. Using this method, quantitative data of chain displacement activity can be obtained, and the chain displacement activity of the recombinase can be accurately analyzed.

[0111] The present invention also provides a kit for detecting the activity of a recombinase, comprising:

[0112] A strand displacement template, wherein the strand displacement template comprises a first nucleic acid molecule and a second nucleic acid molecule that is at least partially complementary to the first nucleic acid molecule, wherein a fluorescent group is disposed at the 5' end of the first nucleic acid molecule, and a quenching group is disposed at the 3' end of the second nucleic acid molecule;

[0113] a strand displacement primer having complementarity to at least a portion of the first nucleic acid molecule; and

[0114] Recombinant enzyme standards.

[0115] In some embodiments, the percentage of complementarity between the first nucleic acid molecule and the second nucleic acid molecule is greater than or equal to 80%; as an example, the percentage of complementarity between the first nucleic acid molecule and the second nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the first nucleic acid molecule and the second nucleic acid molecule are completely complementary.

[0116] In some embodiments, the strand displacement primer has a percent complementarity with the first nucleic acid molecule greater than or equal to 80%; by way of example, the strand displacement primer has a percent complementarity with the first nucleic acid molecule of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the strand displacement primer is completely complementary to the first nucleic acid molecule.

[0117] As an example, the recombinase is selected from bacteriophage T4 Uvs X recombinase, bacteriophage RB69 Uvs X recombinase, bacteriophage Aeh1 Uvs X recombinase, bacteriophage KVP40 Uvs X recombinase, and Escherichia coli RecA recombinase. In some embodiments, in the kit, the molar concentration ratio of the strand displacement primer to the strand displacement template is 1:1 to 1:5.

[0118] In some embodiments, the kit further comprises: at least one of a buffer, magnesium ions, Tris-OAC, and a crowding agent. In some embodiments, the concentration of magnesium ions in the mixed system formed by the kit is 6-20 mmol / L. For example, the concentration of magnesium ions can be 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, and the like.

[0119] In some embodiments, the crowding agent is a macromolecular polymer selected from at least one of PEG, PVP and dextran. In some embodiments, the average molecular weight range of PEG is 4000-10000, and PEG is selected from PEG4000, PEG6000, PEG8000, PEG10000, PEG20000, PEG35000, and PEG100000. In some embodiments, the average molecular weight range of PVP is 10000-60000, and PVP is selected from PVP10000, PVP24000, PVP40000, and PVP55000. In some embodiments, the average molecular weight range of dextran is 10000-70000, and dextran is selected from dextran 10000, dextran 20000, dextran 40000, and dextran 70000.

[0120] In some embodiments, the volume concentration of the crowding agent in the mixed system formed by the kit is 2%-5.5%. For example, the volume concentration of the crowding agent can be 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, etc. The inventors of the present application also found in the experiment that the recombination replacement efficiency can be improved by optimizing the concentration of the crowding agent and the Mg ion.

[0121] The following describes the invention in conjunction with specific embodiments.

[0122] The raw materials and reagents used in the detection method and kit for recombinase activity provided in the embodiments of the present invention can all be purchased from the market.

[0123] Experimental Materials:

[0124] Microplate reader, 200 mmol / L DTT, 10×RPA buffer, 25% crowding reagent, UvsX recombinant enzyme protein (5 mg / ml, 10 U / μL), 280 mmol / L magnesium acetate, 1 M Tris-acetic acid pH 7.9, sterile water, enzyme test dilution buffer.

[0125] The formula of 10×RPA buffer is: 500mmol / L Tris-acetate pH 7.9, 10mmol / L DTT, 2mmol / L dNTPs, 2mg / ml creatine phosphokinase, 500mmol / L creatine phosphokinase sodium salt, 50mmol / L ATP.

[0126] The strand displacement activity test primers are shown in Table 1 below:

[0127] Table 1

[0128]

[0129] Example 1 Preparation of strand displacement substrate

[0130] The strand displacement template primers, P1-FAM and P2-BHQ shown in Table 1 were annealed and incubated in a PCR instrument as strand displacement substrates:

[0131] Table 2

[0132] Strand displacement template primer Volume μl P1-FAM (100 μM) 5 P2-BHQ (100 μM) 5 1×TE Add to 50

[0133] Among them, the annealing incubation program in the PCR instrument is: 98°C, 2 min; 65°C, 1 min; 60°C, 1 min; 55°C, 1 min; 50°C, 1 min; 42°C, 1 min; 25°C, ∞.

[0134] Example 2 Preparation of Standard Test Mixture

[0135] The standard (5 mg / mL) recombinant enzyme with an enzyme activity of 10 U / μL was set at different concentration gradients (0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL) and added to the reaction tubes respectively, and then 2 μL of chain displacement primer P2 was added, and sterile water was added to 10 μL, mixed to prepare a test mixture, and then incubated on ice for 10 minutes.

[0136] Table 3

[0137] Standard Test Mixture Composition Volume (10 μL in total) Standard recombinant enzyme UvsX 0~0.8mg / mL P2 primer (100 μM) 2μL <![CDATA[H 2 The]]> Add to 10.0 μL

[0138] Example 3 Configuration of Active Reaction Buffer

[0139] Prepare the active reaction buffer according to Table 4.

[0140] Table 4

[0141] Active Reaction Buffer Total volume 30 μL 10×RPA buffer 4μL 25% Crowding Reagents 8μL Test substrate prepared in Example 1 2μL 280mmol / L magnesium acetate 2μL 1M tris-acetic acid pH 7.9 2μL 200mmol / L DTT 1.1μL <![CDATA[H 2 The]]> 10.9μL

[0142] Example 4

[0143] 30 μL of the active reaction buffer prepared in Example 3 was added to a 96-well plate, and 5 μL of the standard test mixture (prepared in Example 2) of different concentrations was added to the well plate. After packaging, the plate was slightly shaken to make the bottom of the test wells covered with reagents. Three replicates were set for each sample, and then the sample was quickly placed on an ELISA reader for detection.

[0144] Example 5

[0145] Set the detection program: shake the plate for 1 minute, 37℃ (preheating is required), reaction time for 15 minutes, and detect fluorescence every 30 seconds (excitation: 484nm, emission: 530nm), for a total of 16 minutes. Take the fluorescence value measured within the reaction time of 15 minutes, such as Figure 1 As shown, the slopes of the recombinant enzyme UvsX at each concentration within 2.5 min, 5 min and 5-10 min were statistically analyzed, and the results are shown in Table 5.

[0146] Table 5

[0147] mg / mL Slope (2.5min) Slope (5min) Slope (5-10min) 0 10 9 5.12 0.1 262.4 300.69 181.87 0.2 365.94 391 509.87 0.3 451.71 541.6 769.95 0.4 559.37 724.29 1039.1 0.5 793.31 988.22 1405 0.6 1043.4 1311 1708.9 0.7 1210.4 1515.8 2052.3 0.8 1414 1808.5 2244.3

[0148] The standard curve was drawn with the concentration of the standard recombinant enzyme UvsX in three different time periods (2.5 min, 5 min and 5-10 min) as the horizontal axis and the slope as the vertical axis. The results are shown in Figure 2. Figure 2 The results show that the test data of the standard product test is good, but the reaction is slow. The reactivity of the standard product test can be improved by optimizing the system (magnesium ion concentration, crowding reagent concentration, substrate ratio, incubation test), thereby improving the test efficiency.

[0149] Optimization of strand displacement reaction system

[0150] Example 6 Optimization of magnesium ion concentration

[0151] A reaction system was set up to optimize the magnesium ion concentration (the magnesium ion concentration was within 6-20 mmol / L, and the concentration was adjusted by the added volume in the table below), as shown in Table 6 below:

[0152] Table 6

[0153]

[0154]

[0155] Incubate on ice

[0156]

[0157] The detection was performed using a detection procedure similar to that of Example 5, except that fluorescence (excitation: 484 nm, emission: 530 nm) was detected every 30 seconds for a total of 30 minutes. The fluorescence values ​​measured during the reaction time of each group of reaction systems were plotted into curves (the curves obtained were marked as B2-B10 from low to high magnesium ion working concentrations), as shown in FIG. Figure 3 The slopes of the curves within 2.5 min and 5 min were statistically analyzed, and the results are shown in Table 7.

[0158] Table 7

[0159]

[0160]

[0161] The concentration and slope of magnesium ions in two different time periods (2.5min and 5min) were plotted into a bar graph. The results are as follows: Figure 4 The results show that the difference in the values ​​of magnesium ions at different concentrations is small, and the improvement in the rate of the chain displacement reaction is not significant. For ease of operation, according to the actual reaction system requirements, the subsequent examples will use a magnesium acetate concentration of 10 mmol / L.

[0162] Example 7 Crowding reagent concentration optimization:

[0163] A reaction system was set up to optimize the concentration of the crowding reagent (the volume concentration of the crowding reagent was within 2%-5.5%, and the concentration was adjusted by the added volume in the table below), as shown in Table 8 below:

[0164] Table 8

[0165] Reaction system (40 μl) 1 2 3 4 5 6 7 8 9 P2 (100 μM) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 UvsX (5mg / ml) 0.00 2.40 2.40 2.40 2.40 2.40 2.40 2.40 2.40 <![CDATA[ddH 2 The]]> 8.00 5.60 5.60 5.60 5.60 5.60 5.60 5.60 5.60

[0166] Incubate on ice

[0167]

[0168] The detection was performed using a detection procedure similar to that of Example 5, except that the fluorescence (excitation: 484 nm, emission: 530 nm) was detected every 30 seconds for a total of 30 minutes. The fluorescence values ​​measured during the reaction time of each group of reaction systems were plotted into curves (the obtained curves were marked as C2-C10 from low to high working concentrations of the crowding reagents), as shown in FIG. Figure 5 The slopes of the curves within 2.5 min, 5 min and 10 min were statistically analyzed, and the results are shown in Table 9.

[0169] Table 9

[0170] concentration% Slope (2.5min) Slope (5min) Slope (10min) No enzyme control (C2) 3.6 1.1 1.8545 2(C3) 468 641.82 1195.1 2.5(C4) 563.83 771.65 1430.4 3(C5) 640.97 919.55 1558.3 3.5(C6) 833.71 1168.8 1904.3 4(C7) 852.86 1181 1625.5 4.5(C8) 496.74 510.05 669.27 5(C9) 332.4 381.98 553.22 5.5(C10) 305.49 372.16 526.49

[0171] The concentration and slope of the crowding reagent in three different time periods (2.5min, 5min and 10min) were plotted into a bar graph. The results are as follows: Figure 6 As shown. The results show that: in the range of 2-3.5% crowding reagent concentration, as the crowding reagent concentration increases, the slope increases first; when the crowding reagent concentration reaches 3.5%, the slope begins to decline. It can be seen that when the crowding reagent concentration in the UvsX activity test system is 3.5%, it can effectively increase the chain displacement reaction rate, and the subsequent test uses a crowding reagent concentration of 3.5%.

[0172] Example 8 Optimization of the concentration ratio of strand displacement template to strand displacement primer

[0173] Set up a reaction system to optimize the concentration of the strand displacement primer (P2) (the concentration is adjusted by the added volume in the table below), as shown in Table 10 below:

[0174] Table 10

[0175] Reaction system (40 μl) 1 2 3 4 5 6 7 8 9 10 11 P2 (100 μmol / L) 2.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 UvsX (5mg / ml) 0.00 2.40 2.40 2.40 2.40 2.40 2.40 2.40 2.40 2.40 2.40 <![CDATA[ddH 2 The]]> 8.00 7.40 7.20 7.00 6.80 6.60 6.40 6.20 6.00 5.80 5.60

[0176] Incubate on ice

[0177]

[0178]

[0179] The detection was performed using a detection procedure similar to that of Example 5, except that the fluorescence (excitation: 484 nm, emission: 530 nm) was detected every 30 seconds for a total of 30 minutes. The fluorescence values ​​measured during the reaction time of each group of reaction systems were plotted into curves (the curves obtained were marked as E1-E11 from low to high molar ratios). The results are shown in FIG. Figure 7 The slopes of the curves within 2.5 min, 5 min and 5 min were calculated, and the results are shown in Table 10.

[0180] Table 11

[0181]

[0182]

[0183] The molar ratio and slope of the strand displacement template and strand displacement primer in 5 different time periods (0-1min, 1-2min, 2-3min, 3-4min and 4-5min) were plotted into a bar graph. The results are shown in Figure 8 As shown. The results show that: in the range of 1:1 to 1:3, the slope increases with the increase of the concentration of the strand displacement primer; when the molar ratio of the strand displacement template to the strand displacement primer is 1:4 to 1:6, the slope decreases slightly, but is basically stable; when the molar ratio of the strand displacement template to the strand displacement primer is 1:7 to 1:10, the slope decreases slowly. It can be seen that the strand displacement reaction rate can be effectively increased when the molar ratio of the strand displacement template to the strand displacement primer is 1:3.

[0184] Example 9 Configuration Optimized Strand Displacement Reaction System

[0185] Prepare the reaction system 1 containing the recombinant enzyme standard in a 200 μL tube according to the component system determined in Table 12 below:

[0186] Table 12

[0187]

[0188] According to the component system determined in Table 13 below, prepare the reaction system 2 containing the recombinant enzyme to be tested in a 200 μL tube:

[0189] Table 13

[0190]

[0191]

[0192] Reaction system 3 was prepared as a blank control by removing the recombinant enzyme standard in Table 12.

[0193] After thoroughly mixing reaction system 1, reaction system 2, and reaction system 3, respectively draw the reaction solution into a 96-well plate, shake the plate slightly to make the bottom of the detection wells covered with reagents, set 3 replicates for each sample, and then quickly place it on the microplate reader for detection. The specific operating procedures are as follows: shake the 96-well plate for 1 minute, 37°C (need to be preheated in advance) for a reaction time of 15 minutes, and detect fluorescence every 30 seconds (excitation: 484nm, emission: 530nm), for a total of 16 minutes. Take the fluorescence value within 0-2min for calculation, and count the absolute value of the fluorescence value change per unit time of three parallel tests (abbreviated as reaction rate in the table) and its average value (abbreviated as the average value of reaction rate in the table), and analyze the experimental results as shown in Table 14:

[0194] Table 14

[0195]

[0196] With the amount of enzyme per unit volume of the recombinant enzyme standard as the horizontal axis and the reaction rate as the vertical axis, a standard curve graph of the two is drawn, as shown in Fig. 9 As shown, the linear equation y = 117.7x-36.733 is obtained, and the correlation coefficient R 2 =0.9956. Substituting the reaction rate value of the recombinase to be tested into the above equation, the relative activity result of the test product can be obtained. After calculation, the input enzyme amount of the recombinase to be tested is 9U. According to the concentration of the reaction system, the strand displacement activity of the recombinase to be tested is converted to 11.2U / μL.

[0197] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting recombinase activity, characterized in that: The steps include: (1) obtaining a strand displacement template, wherein the strand displacement template comprises a first nucleic acid molecule and a second nucleic acid molecule that is at least partially complementary to the first nucleic acid molecule, wherein a fluorescent group is disposed at the 5' end of the first nucleic acid molecule and a quenching group is disposed at the 3' end of the second nucleic acid molecule; (2) mixing a recombinase standard, a strand displacement primer and the strand displacement template, and detecting the fluorescence value of the mixed system within a given period of time, wherein the strand displacement primer is complementary to at least a portion of the first nucleic acid molecule; (3) establishing a linear fitting equation y=ax+b of the absolute value of the first fluorescence value change per unit time and the enzyme activity based on the change of the fluorescence value, wherein y is the absolute value of the fluorescence value change, x is the enzyme concentration or enzyme activity, and a and b are constants; (4) Determine the activity of the recombinant enzyme to be tested based on the linear fitting equation.

2. The detection method according to claim 1, characterized in that Determining the activity of the recombinase to be tested comprises: The recombinase to be tested is substituted for the recombinase standard, and the fluorescence value within a given time period is obtained according to the method of step (1) and step (2); Based on the change in the fluorescence value, determining an absolute value of a change in a second fluorescence value per unit time; Determining the activity of the recombinase to be tested based on the absolute value of the change in the second fluorescence value and the linear fitting equation y=ax+b; Optionally, when the recombinase to be tested includes two or more recombinases, Determining the activity of the recombinase to be tested comprises: Respectively obtaining the absolute value of the change in the second fluorescence value of two or more recombinases to be tested with the same concentration within a unit time; Comparing the absolute values ​​of the changes in the second fluorescence values ​​of two or more recombinases to be tested; Based on the comparison results, the activity ranking of the two or more recombinases to be tested is determined.

3. The detection method according to any one of claims 1 to 2, characterized in that: The given time period is within 5-10 minutes after mixing the recombinase standard, the strand displacement primer and the strand displacement template.

4. The detection method according to any one of claims 1 to 3, characterized in that: The mixed recombinase, the strand displacement primer and the strand displacement template include: mixing the recombinase and the strand displacement primer, and incubating to obtain a mixture; mixing the mixture with the strand displacement template; Optionally, the incubation time is 0-10 min; Optionally, the unit time is 20-40s; and / or The given time period is within 5-10 minutes after mixing the mixture with the strand displacement template; Optionally, the method for detecting the fluorescence value within a given time period is: detecting the fluorescence value once every 20-35 seconds, taking the previous N detection results, where N is an integer of 5 to 20.

5. The detection method according to any one of claims 1 to 4, characterized in that: The complementarity percentage between the first nucleic acid molecule and the second nucleic acid molecule is greater than or equal to 80%; Optionally, the percentage of complementarity between the first nucleic acid molecule and the second nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the first nucleic acid molecule and the second nucleic acid molecule are completely complementary; Optionally, the complementarity percentage between the strand displacement primer and the first nucleic acid molecule is greater than or equal to 80%; Optionally, the strand displacement primer is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the first nucleic acid molecule, or the strand displacement primer is completely complementary to the first nucleic acid molecule.

6. The detection method according to any one of claims 1 to 5, characterized in that: The recombinase is selected from bacteriophage T4 UvsX recombinase, bacteriophage RB69 Uvs X recombinase, bacteriophage Aeh1 Uvs X recombinase, bacteriophage KVP40 Uvs X recombinase, and Escherichia coli RecA recombinase; Optionally, the step of mixing the recombinase standard, the strand displacement primer and the strand displacement template comprises mixing the strand displacement primer and the strand displacement template at a molar concentration ratio of 1:1 to 1:5; Optionally, the mixed system further comprises: at least one of a buffer, magnesium ions, Tris-OAC or a crowding agent; Optionally, in the mixed system, the concentration of magnesium ions is 6-20 mmol / L; Optionally, the crowding agent is selected from at least one of PEG, PVP and dextran; Optionally, the average molecular weight of the PEG is in the range of 4000-10000; The average molecular weight of the PVP is in the range of 10000-60000; The average molecular weight of the dextran is in the range of 10000-70000; Optionally, the volume concentration of the crowding agent is 2%-5.5%.

7. The detection method according to any one of claims 1 to 6, characterized in that: The quenching group is BHQ, the fluorescent group is FAM, 484 nm is used as the excitation light, and 530 nm is used as the emission light to detect the fluorescence value within a given period of time.

8. A kit, characterized in that: The kit is used to detect the activity of the recombinase, and comprises: A strand displacement template, the strand displacement template comprising a first nucleic acid molecule and a second nucleic acid molecule that is at least partially complementary to the first nucleic acid molecule, the 5' end of the first nucleic acid molecule is provided with a fluorescent group, and the 3' end of the second nucleic acid molecule is provided with a quenching group; a strand displacement primer having complementarity to at least a portion of the first nucleic acid molecule; and a recombinase standard.

9. The kit according to claim 8, characterized in that The complementarity percentage between the first nucleic acid molecule and the second nucleic acid molecule is greater than or equal to 80%; Optionally, the percentage of complementarity between the first nucleic acid molecule and the second nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or the first nucleic acid molecule and the second nucleic acid molecule are completely complementary; Optionally, the complementarity percentage between the strand displacement primer and the first nucleic acid molecule is greater than or equal to 80%; Optionally, the strand displacement primer is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the first nucleic acid molecule, or the strand displacement primer is completely complementary to the first nucleic acid molecule.

10. The kit according to any one of claims 8 to 9, characterized in that The recombinase is selected from bacteriophage T4 UvsX recombinase, bacteriophage RB69 Uvs X recombinase, bacteriophage Aeh1 Uvs X recombinase, bacteriophage KVP40 Uvs X recombinase, and Escherichia coli RecA recombinase; Optionally, in the kit, the molar concentration ratio of the strand displacement primer to the strand displacement template is 1:1 to 1:5; Optionally, the kit further comprises: at least one of a buffer, magnesium ions, Tris-OAC, and a crowding reagent; Optionally, the concentration of the magnesium ions in the mixed system formed by the kit is 6-20 mmol / L; Optionally, the crowding agent is selected from at least one of PEG, PVP and dextran; Optionally, the average molecular weight of the PEG is in the range of 4000-10000; The average molecular weight of the PVP is in the range of 10000-60000; The average molecular weight of the dextran is in the range of 10000-70000; Optionally, the volume concentration of the crowding agent in the mixed system formed by the kit is 2%-5.5%.

Images