Hairpin template for detecting Bst DNA polymerase activity and application, kit and method thereof

Abstract

The invention discloses a hairpin template for detecting Bst DNA polymerase activity and application, a kit and a method thereof, and relates to the technical field of enzyme activity detection, and the nucleotide sequence of the hairpin template is as shown in SEQ ID No.1. The invention provides a specific hairpin template. The hairpin template has a stem-loop structure, is in a single-chain closed state when not amplified, is less combined with an embedded fluorescent nucleic acid dye, and has a lower background signal; when the Bst DNA polymerase is added, the enzyme has 5 '-> 3' DNA polymerase activity and strong strand displacement activity, the hairpin template can be self-triggered to amplify under the condition of no primer, the hairpin template is expanded and copied to form double-stranded DNA under the action of the Bst DNA polymerase, and the embedded fluorescent nucleic acid dye can be specifically combined with the double-stranded DNA to enhance a fluorescent signal of a system. The activity of the Bst DNA polymerase can be calculated according to the relationship between the double-stranded DNA and the fluorescence signal intensity.

Description

Technical Field

[0001] This invention relates to the field of enzyme activity detection technology, and in particular to a hairpin template for detecting Bst DNA polymerase activity, its application, kit, and method. Background Technology

[0002] Bst DNA polymerase is a homolog of the large fragment of DNA polymerase I from Bacillus stearothermophilus (Bst DNA polymerase, large fragment). It possesses 5′→3′ DNA polymerase activity and strong strand substitution activity, but lacks 5′→3′ exonuclease activity. It is a commonly used thermostable DNA polymerase. Bst DNA polymerase is widely used in isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and molecular diagnostics. Its activity detection is a key step in ensuring enzyme quality and application performance.

[0003] Currently, the mainstream methods for detecting Bst DNA polymerase activity and their application status are as follows: 1) Radiolabeling method: by incorporating radiolabeled dNTPs (such as... 32 1) Polymerase activity assay (P-dCTP): High sensitivity, but safety and waste disposal issues exist; 2) Fluorescent labeling: Fluorescently labeled primers / dNTPs are used to detect amplified products through real-time fluorescence signals. The operation is simple, but the cost of fluorescent labeling is high; 3) Colorimetric method: For example, enzyme-coupled reactions based on pyrophosphate release indirectly reflect activity through changes in absorbance. Suitable for high throughput, but with low sensitivity; 4) Gel electrophoresis: Quantifying activity through electrophoretic bands of amplified products. Intuitive, but time-consuming and not suitable for high throughput; 5) Biosensor technology: Emerging nanomaterials or electrochemical sensors (such as graphene electrodes) can directly detect polymerase activity, but are still in the research stage; 6) Commercial kits: Several companies (such as NEB, Thermo Fisher, and Takara) provide standardized activity assay kits, usually based on fluorescence or colorimetric principles, but they are expensive and dependent on specialized equipment.

[0004] However, the mainstream method for detecting the activity of Bst DNA polymerase has the following disadvantages: 1) sensitivity and specificity are contradictory to each other: high sensitivity methods such as radioactive labeling have safety risks, and safe methods such as colorimetry may not be able to detect low activity samples; 2) many interference factors: such as the current fluorescence method mostly uses double-stranded circular plasmid DNA as a template, but due to the strong strand displacement activity of Bst DNA polymerase, the fluorescence amount is easily disturbed, and the reaction stability is poor; 3) time-consuming and complex: gel electrophoresis and radioactive methods are complicated and not suitable for rapid detection, and commercial kits usually require specific enzyme markers or qPCR instruments, which limits the application at the grassroots level; 4) lack of standardization: different manufacturers have different definitions of activity units, making it difficult to compare results horizontally; 5) high cost: high-end methods such as fluorescence labeling are highly dependent on equipment, which is not friendly to small-scale laboratories; 6) difficulty in real-time monitoring: existing technologies are difficult to track the dynamic changes of polymerase activity in real time, such as activity decay during the reaction. Therefore, although the current Bst DNA polymerase activity detection technology is diversified, it still faces core problems such as sensitivity, stability, standardization, and cost. SUMMARY

[0005] The main purpose of the present application is to provide a hairpin template for detecting the activity of Bst DNA polymerase and its application, kit and method, aiming to provide a method for detecting the activity of Bst DNA polymerase more quickly and accurately.

[0006] To achieve the above-mentioned purpose, the present application provides a hairpin template for detecting the activity of Bst DNA polymerase, and the nucleotide sequence of the hairpin template is shown as SEQ ID No. 1.

[0007] The present application also provides an application of the aforementioned hairpin template for detecting the activity of Bst DNA polymerase in preparing a product for detecting the activity of Bst DNA polymerase.

[0008] The present application also provides a kit for detecting the activity of Bst DNA polymerase, comprising a PCR reaction solution and a DNA standard solution;

[0009] The PCR reaction solution comprises an embedded fluorescent nucleic acid marker, a buffer, dNTP, and the aforementioned hairpin template for detecting the activity of Bst DNA polymerase;

[0010] The DNA standard solution comprises an embedded fluorescent nucleic acid marker, a buffer, and a DNA standard.

[0011] The present application also provides a method for detecting the activity of Bst DNA polymerase, comprising the following steps:

[0012] S1, provide a Bst DNA polymerase to be tested and the kit for detecting the Bst DNA polymerase activity in the foregoing;

[0013] S2, gradient dilution is carried out on DNA standard sample reaction liquid, a plurality of gradient concentrations of DNA standard sample reaction liquid are obtained, the plurality of gradient concentrations of DNA standard sample reaction liquid are subjected to first isothermal amplification treatment, and the fluorescence value of the plurality of gradient concentrations of DNA standard sample reaction liquid is obtained, and a standard curve formula is prepared through the DNA amount in the plurality of gradient concentrations of DNA standard sample reaction liquid and the corresponding fluorescence value;

[0014] S3, a Bst DNA polymerase to be tested is provided, the Bst DNA polymerase to be tested is mixed with PCR reaction liquid, and then subjected to second isothermal amplification treatment, and the end-point fluorescence value is obtained, the end-point fluorescence value is substituted into the standard curve formula, and the amount of double-stranded DNA newly generated in the second isothermal amplification treatment process is obtained.

[0015] S4, the Bst DNA polymerase activity is calculated according to the amount of double-stranded DNA newly generated in the second isothermal amplification treatment process.

[0016] In the technical scheme of the present application, a specific hairpin template is provided, the hairpin template has a stem-loop structure, and is in a single-stranded closed state when not amplified, has less combination with an embedded fluorescent nucleic acid dye, and has a lower background signal; when the Bst DNA polymerase is added, the enzyme has 5'→3' DNA polymerase activity and strong strand displacement activity, the hairpin template can be self-triggered to amplify under the condition that there is no primer, is unfolded and copied to form double-stranded DNA under the action of the Bst DNA polymerase, the embedded fluorescent nucleic acid dye can specifically combine with the double-stranded DNA to enhance the fluorescence signal of the system, and the Bst DNA polymerase activity can be calculated according to the relationship between the double-stranded DNA and the fluorescence signal intensity. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical scheme in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained according to the structures shown in the drawings without creative labor for those skilled in the art.

[0018] Figure 1 It is a schematic diagram of the secondary structure of the hairpin template in the present application;

[0019] Figure 2 It is a relationship diagram of different concentrations of DNA standard samples and corresponding fluorescence values in embodiment 1 of the present application;

[0020] Figure 3The standard curve fitting chart in Example 1 of the present application;

[0021] Figure 4 The real-time fluorescence value monitoring result chart of the Bst DNA polymerase isothermal amplification reaction system in Example 2 of the present application; The real-time fluorescence value monitoring result chart of the Bst DNA polymerase isothermal amplification reaction system in Example 2 of the present application;

[0022] Figure 5 The real-time fluorescence value monitoring result chart of the Bst DNA polymerase isothermal amplification reaction system in Example 2 of the present application;

[0023] The implementation, functional features and advantages of the present application will be further described with reference to the accompanying drawings. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments are not specified by the manufacturers, they are all conventional products that can be purchased in the market. In addition, the meaning of "and / or" in the whole text includes three parallel solutions. For example, "A and / or B" includes the A solution, or the B solution, or the solution of A and B at the same time. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that the technical solutions can be realized by the ordinary skilled in the art. When the combination of the technical solutions appears to be contradictory or unachievable, it should be considered that the combination of the technical solutions does not exist and is not within the protection scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without making creative efforts are within the protection scope of the present application.

[0025] Therefore, the present application provides a hairpin template for detecting the activity of Bst DNA polymerase, and the nucleotide sequence of the hairpin template is shown as SEQ ID No. 1.

[0026] In the technical solutions of the present application, the present application provides a specific hairpin template, which has a stem-loop structure and is in a single-stranded closed state without amplification, has less combination with the embedded fluorescent nucleic acid dye, and has a lower background signal. When the Bst DNA polymerase is added, the enzyme has 5'→3' DNA polymerase activity and strong strand displacement activity. Under the condition without primer, the hairpin template can be self-triggered to amplify, and under the action of the Bst DNA polymerase, the hairpin template is unfolded and replicated to form double-stranded DNA. The embedded fluorescent nucleic acid dye can specifically combine with the double-stranded DNA to enhance the fluorescence signal of the system. According to the relationship between the double-stranded DNA and the fluorescence signal intensity, the activity of the Bst DNA polymerase can be calculated.

[0027] It can be understood that the hairpin template is a secondary structure formed by folding a single-stranded DNA (ssDNA) itself, which includes a stem and a loop, the stem is a double-stranded region formed by hydrogen bonding of complementary paired bases, and the loop is a single-stranded region in the middle which does not participate in pairing, and a structural schematic diagram is as shown in Figure 1 .

[0028] It can be understood that the hairpin template of the present application has the following advantages:

[0029] (1) Self-triggered amplification, less interference: The stem-loop region of the hairpin template can be used as a primer binding site, so that no external primer is needed, the reaction system is simplified, and the interference of dimers in the amplification process is reduced.

[0030] (2) Low false positive rate: The closed state of the hairpin template of the present application can prevent random annealing of the template, thereby reducing the probability of false positives.

[0031] (3) Signal amplification effect: When the hairpin template is unfolded and replicated under the action of Bst DNA polymerase to form double-stranded DNA (dsDNA), the embedded fluorescent nucleic acid dye specifically binds to dsDNA, and the fluorescence signal is significantly enhanced (up to 10 3 ~ 10 4 times), greatly improving the detection sensitivity (as low as amol level activity). Compared with linear templates, the hairpin structure produces more dsDNA through "folding-unfolding" cycles, and the signal accumulates faster.

[0032] (4) Low background noise: The hairpin structure remains in a closed state (single-stranded) when not amplified, and the fluorescent dye binds very little, resulting in low initial background signal; only when the polymerase activity triggers amplification, the fluorescence significantly increases, and the signal-to-noise ratio (S / N) is better than that of linear templates.

[0033] (5) Fast response and real-time monitoring: Traditional PCR requires repeated denaturation-annealing, while the hairpin structure can spontaneously unwind-reanneal at 65°C under isothermal conditions, achieving real-time signal output, suitable for LAMP and other isothermal amplification techniques.

[0034] (6) No labeling and low cost: Directly use the universal binding property of dye and dsDNA, without the need to synthesize fluorescently labeled primers or dNTPs (such as FAM-dUTP), which reduces costs and avoids the influence of labeling efficiency fluctuations. The same dye can be adapted to different hairpin template designs, without the need to customize probes for each template.

[0035] The present application also provides a use of the aforementioned hairpin template for detecting Bst DNA polymerase activity in the preparation of a product for detecting Bst DNA polymerase activity. It can be understood that the product for detecting Bst DNA polymerase activity includes kits, reagents, test papers, etc., which are convenient to use in different scenarios.

[0036] The application further provides a kit for detecting Bst DNA polymerase activity, comprising a PCR reaction solution and a DNA standard solution.

[0037] The PCR reaction solution comprises an embedded fluorescent nucleic acid marker, a buffer, dNTPs, and the hairpin template for detecting Bst DNA polymerase activity mentioned above.

[0038] The DNA standard solution comprises an embedded fluorescent nucleic acid marker, a buffer, and a DNA standard.

[0039] In the technical solution of the application, each substance in the PCR reaction solution can be independently packaged or mixedly packaged under the condition of not affecting the amplification process. Each substance in the DNA standard solution can be independently packaged and prepared on demand when used. It can be understood that the DNA standard solution is used as a standard substance, that is, the corresponding relationship between the amount of double-stranded DNA newly generated from the hairpin template in the isothermal amplification process and the increased fluorescence value is obtained through the corresponding relationship between the DNA standard solution and the fluorescence value. It should be noted that the fluorescence value is the net fluorescence value after deducting the background fluorescence.

[0040] In some embodiments of the application, the embedded fluorescent nucleic acid marker comprises SYBR Green fluorescent nucleic acid dye or EvaGreen fluorescent nucleic acid dye; and / or the dNTPs comprise dATP, dTTP, dCTP, and dGTP; and / or the DNA standard comprises a Lambda DNA standard.

[0041] It can be understood that the traditional P labeling method has radiation hazards and requires special protection and waste treatment. 32 The application uses fluorescent dye SYBR Green, which has no radiation risk and is relatively low in cost.

[0042] In some embodiments of the application, in the PCR reaction solution: the system final concentration of the hairpin template for detecting Bst DNA polymerase activity is 0.25-0.4 μM; and / or the dNTPs comprise dATP, dTTP, dCTP, and dGTP, and the system final concentration of each of the dATP, dTTP, dCTP, and dGTP is 0.2-0.25 mM. Controlling the system final concentration of the hairpin template and the dNTPs in the above range can ensure that the amplification speed of the hairpin template is relatively fast, and the detection fluorescence intensity is easy to measure.

[0043] The application further provides a method for detecting Bst DNA polymerase activity, comprising the following steps:

[0044] Step S1, providing a Bst DNA polymerase to be tested and the aforementioned kit for detecting the activity of the Bst DNA polymerase;

[0045] Step S2, gradient dilution is performed on the DNA standard reaction solution to obtain a plurality of DNA standard reaction solutions with gradient concentrations; the plurality of DNA standard reaction solutions with gradient concentrations are subjected to first isothermal amplification treatment to obtain fluorescence values of the plurality of DNA standard reaction solutions with gradient concentrations; and a standard curve formula is made by the DNA amounts in the plurality of DNA standard reaction solutions with gradient concentrations and the corresponding fluorescence values;

[0046] Step S3, providing a Bst DNA polymerase to be tested, mixing the Bst DNA polymerase to be tested with a PCR reaction solution, and then performing second isothermal amplification treatment to obtain an end-point fluorescence value; and substituting the end-point fluorescence value into the standard curve formula to obtain the amount of newly generated double-stranded DNA in the second isothermal amplification treatment process.

[0047] Step S4, calculating the activity of the Bst DNA polymerase according to the amount of newly generated double-stranded DNA in the second isothermal amplification treatment process.

[0048] The principle of the method for detecting the activity of the Bst DNA polymerase is as follows: (1) the Bst DNA polymerase has 5'→3' DNA polymerase activity and strong strand displacement activity, but lacks 5'→3' exonuclease activity, and is a commonly used heat-stable DNA polymerase. When a hairpin structure template is used as a substrate and the Bst DNA polymerase is added for amplification reaction, new double-stranded DNA can be generated; (2) SYBR Green and other intercalating fluorescent nucleic acid dyes have no sequence specificity and can intercalate into the minor groove of the newly generated double-stranded DNA; (3) when not combined with DNA, the fluorescence signal of SYBR Green and other intercalating fluorescent nucleic acid dyes is extremely weak (low background and small interference), and once intercalated into the newly generated dsDNA (for example, base pairs), the fluorescence intensity is significantly enhanced (about 1000 times), which can significantly amplify the fluorescence signal and improve the detection sensitivity; (4) in a real-time fluorescent quantitative PCR instrument, the excitation wavelength is set to 497 nm and the emission wavelength is set to 520 nm, so that the amount of amplification product dsDNA can be indirectly reflected by detecting the fluorescence intensity of each cycle; (5) the standard curve formula made according to the standard dsDNA and net fluorescence value can accurately calculate the amount of newly generated dsDNA and obtain the dNTP consumption, so as to obtain the activity of the Bst DNA polymerase according to the definition. The definition of the activity of the national standard Bst DNA polymerase is that the amount of enzyme required for synthesizing 1.29 nmol of deoxynucleotide into double-stranded DNA within 1 min at 65℃ is 1 activity unit (U).

[0049] Specifically:

[0050] Step S2, dsDNA standard curve preparation:

[0051] A series of gradient concentrations of dsDNA standard reaction solution was provided, and first isothermal amplification treatment was performed to obtain the fluorescence value of each gradient concentration of dsDNA standard reaction solution. Each fluorescence value was deducted from the background fluorescence to obtain the net fluorescence value of each gradient concentration of dsDNA standard reaction solution (in multiple repeated experiments, the average value of the net fluorescence values of multiple repetitions can be used as the final net fluorescence value, and the standard deviation of multiple repetitions can be used as the error value). According to the net fluorescence value of each gradient concentration of dsDNA standard reaction solution and the corresponding dsDNA standard concentration in the dsDNA standard reaction solution, a standard curve was prepared (as shown in formula 1).

[0052] y = A + Bx (formula 1);

[0053] In the formula: A is the intercept of the linear equation fitting curve; B is the slope of the linear equation fitting curve; y is the net fluorescence value; x is the amount of double-stranded DNA, in nanograms (ng).

[0054] Preferably, in step S2, the first isothermal amplification treatment conditions are: constant temperature at 60-65°C for 90-100 cycles, and each cycle time is 14-20s.

[0055] Step S3, calculation of the amount of newly generated double-stranded DNA in the polymerization reaction

[0056] (1) The Bst DNA polymerase to be tested was provided, and the Bst DNA polymerase to be tested and the PCR reaction solution were mixed to perform second isothermal amplification treatment to obtain the fluorescence data of the last cycle (end point fluorescence value) in the second isothermal amplification treatment. The end point fluorescence value is deducted from the fluorescence value of the blank control to obtain the net fluorescence value (y1). The net fluorescence value is substituted into formula 1 to calculate the amount of newly generated double-stranded DNA (x1) in the polymerization reaction.

[0057] Preferably, in step S3, the second isothermal amplification treatment conditions are: constant temperature at 60-65°C for 30-50 cycles, and each cycle time is 14-20s.

[0058] Step S4, Bst DNA polymerase activity calculation:

[0059] (1) dNTP consumption calculation:

[0060] The dNTP consumption is represented by y2, in nmol, and is calculated according to formula 2:

[0061] y2 = x1 / (2 x 324.5) (formula 2);

[0062] In the formula: x1 is the amount of newly generated double-stranded DNA, in nanograms (ng); 2 is the double-stranded DNA (composed of 2 single-stranded DNAs); 324.5 is the relative average molecular mass of dNTPs.

[0063] (2) Calculation of Bst DNA polymerase activity:

[0064] According to the definition of DNA polymerase activity units, the enzyme activity of Bst DNA polymerase is expressed as S, with units of U / μL. The dNTP consumption within the range of the standard curve (Equation 1) is calculated according to Equation 3:

[0065] S=(y2-0.003)×D / 1.77 (Formula 3);

[0066] In the formula: y2 is the amount of dNTP consumed, in nanomoles (nmol); D is the dilution factor of the enzyme.

[0067] Understandably, if there is no dNTP consumption within the standard curve range, the Bst DNA polymerase to be tested can be diluted and steps S1-S4 can be performed until its dNTP consumption is within the standard curve range.

[0068] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0069] Experimental materials:

[0070] 1. Instruments and equipment: Real-time PCR instrument

[0071] 2. Reagents and consumables:

[0072] (1) The hairpin template sequence is shown in SEQ ID No. 1 (the secondary structure of the hairpin template is as follows). Figure 1 As shown):

[0073] 5'-TCAAGTCGTCAGAAATACATGACCACtagcgaaACCAgtgaacctaatcccTGCTCCCGCGGCCGatctgcCGGCCGCGGG-3'.

[0074] (2) Fluorescent nucleic acid dye SYBR Green;

[0075] (3) Lamda DNA standard: 350 ng / μL;

[0076] (4) dNTP (deoxynucleotide triphosphate): including dATP (deoxyadenosine triphosphate), dTTP (deoxythymidine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), each 25 mmol / L;

[0077] (5) MgCl2: 25 mmol / L;

[0078] (6) 10x PCR Buffer: 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, pH 8.3 at room temperature;

[0079] (7) 10x Isothermal Amplification Buffer: 200 mM Tris-HCl, 100 mM (NH4)2SO4, 500 mM KCl, 20 mM MgSO4, 1% Tween-20, pH 8.8 at room temperature;

[0080] (8) Enzyme diluent: 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, 0.1% X-100, pH 7.1 at room temperature.

[0081] Example 1: Preparation of Lambda DNA standard curve

[0082] Step 1: After diluting the Lambda DNA (350 ng / μL) standard to 100 ng / μL, sequentially dilute to 50 ng / μL, 25 ng / μL, 12.5 ng / μL, 6.25 ng / μL, 3.125 ng / μL, 1.5625 ng / μL, 0.78125 ng / μL, 0 ng / μL. Configure the standard detection system of different concentrations according to the following Table 1, and make 3 repeats for each gradient;

[0083] Table 1 Standard detection system

[0084] Component Volume (μL) 10 x PCR Buffer 2 20 x SYBR Green fluorescent nucleic acid dye 1 Lambda DNA standard 1 H2O 16 Total 20

[0085] Step 2: After mixing the 8 groups of standard detection systems configured according to Table 1, place them in the fluorescence quantitative PCR instrument at 65°C, 16s, and cycle 99 times (constant temperature reaction), and monitor the fluorescence signal (excitation / emission wavelength: 497 nm / 520 nm) in FAM channel in real time;

[0086] Step 3: Select the original curve data at the 3rd minute for processing, and the relationship between fluorescence and DNA standard input amount is shown in Figure 2 ​​

[0087] Step 4: Open the data using the software of the fluorescence PCR instrument, and perform calculations according to the following steps and formula (A.1):

[0088] a) Select the endpoint fluorescence values ​​(plateau phase values) at the 3rd minute of the 6 groups of Lambda DNA standard detection systems and background control groups (groups without added standards) from the exported data file. Subtract the average fluorescence value of the background control group from the fluorescence value of each replicate of each gradient Lambda DNA standard detection system to obtain the net fluorescence value of each replicate of each gradient Lambda DNA standard detection system. Then calculate the average net fluorescence value of the Lambda DNA standard detection system at each gradient (results are shown in Table 2) and the standard deviation (SD). It should be noted that the standard curve obtained from the selected 6 Lambda DNA standard concentrations has good linearity. Therefore, 0–12.5 ng / μL standards were finally selected to construct the standard curve. The amount of Lambda DNA standard in Table 2 refers to the mass of Lambda DNA standard in the Lambda DNA standard detection system.

[0089] Table 2 Net fluorescence values ​​of the Lambda DNA standard detection systems at different gradients

[0090] Lambda DNA standard amount (ng) Mean of net fluorescence values 0 0 0.78125 253.8866667 1.5625 487.1 3.125 1029.413333 6.25 1970.92 12.5 3453.07

[0091] b) Using the amount of DNA (ng) in the Lambda DNA standard as the x-axis and the average net fluorescence value of the Lambda DNA standard detection system at each gradient as the y-axis, with the standard deviation (SD) as the error value, a polynomial fitting analysis was performed to construct a working curve, from which a standard curve between the average net fluorescence value and the double-stranded DNA concentration was obtained. Figure 3 As shown in equation A.1 below, its R 2 ≥0.98.

[0092] y = 277.41x + 79.316(R) 2 =0.9942)(Equation A.1);

[0093] In the formula: y is the average net fluorescence value; x is the amount of double-stranded DNA, in nanograms (ng). Example 2: Commercial Bst DNA Polymerase (NEB) Activity Assay

[0094] 1. Calculation of the amount of newly generated double-stranded DNA in the polymerization reaction

[0095] Step 1: Label commercially available enzyme with Bst at an activity of 8 U / μL DNA Polymerase (NEB) was diluted to 1 U / μL, and then to 0.05 U / μL, 0.03 U / μL, 0.02 U / μL, 0.01 U / μL, 0.006 U / μL, 0.003 U / μL, 0.001 U / μL, respectively. The isothermal amplification reaction system was prepared as shown in Table 3, and 7 groups of Bst DNA Polymerase isothermal amplification reaction system, while setting a negative control group without enzyme, vortex mixing for 8 s, micro centrifuge for 3 s, 3 replicates for each treatment;

[0096] Table 3 Isothermal amplification reaction system

[0097]

[0098] Step 2: The 8 groups of isothermal amplification reaction system prepared above were placed in a fluorescence quantitative PCR instrument, and cycled 40 times (constant temperature reaction) at 65°C, 16 s, and the fluorescence signal (excitation / emission wavelength: 497 nm / 520 nm) was monitored in real time in the FAM channel. The fluorescence amount change is shown in Figure 4

[0099] Step 3, export data file, select the data file in each gradient Bst DNA Polymerase isothermal amplification reaction system end point fluorescence data of the last cycle. First, calculate the average fluorescence value of the negative control group with 3 replicates, and then subtract the average fluorescence value of the negative control group from each gradient Bst DNA Polymerase isothermal amplification reaction system each repeat fluorescence value minus the average fluorescence value of the negative control group, to get each gradient Bst DNA Polymerase isothermal amplification reaction system each repeat fluorescence value, then calculate the amount of newly generated double-stranded DNA (x1) under each gradient Bst

[0100] DNA Polymerase isothermal amplification reaction system average value of the net fluorescence value (y1). The amount of newly generated double-stranded DNA (x1) under each gradient Bst DNA Polymerase isothermal amplification reaction system average value y1 of the net fluorescence value is substituted into formula (A.1) to calculate the amount of newly generated double-stranded DNA (x1); the results are shown in Table 4;

[0101] 2, the amount of Bst DNA Polymerase isothermal amplification reaction system dNTP consumption calculation

[0102] The amount of Bst DNA Polymerase isothermal amplification reaction system dNTP consumption is represented by y2, with units of nmol, calculated according to formula (A.2):

[0103] ​y2 = x1 / (2 x 324.5) (Formula A.2);

[0104] In the formula: x1 is the amount of newly generated double-stranded DNA, in nanograms (ng); 2 is double-stranded DNA (consisting of 2 single-stranded DNAs); 324.5 is the relative average molecular weight of dNTP;

[0105] The results are shown in Table 4.

[0106] 3, Bst Enzymatic activity calculation of DNA polymerase

[0107] According to the definition of DNA polymerase activity unit, Bst The enzymatic activity of DNA polymerase is represented by S, in units of U / μL. The dNTP consumption amount selected in the range of 0.0077 nmol to 0.281 nmol (the range corresponding to the calibration curve in Example 1) is calculated according to Formula (A.3):

[0108] S = (y2-0.003) x D / 1.77 (Formula A.3);

[0109] In the formula: y2 is the dNTP consumption amount, in nanomoles (nmol); D is the dilution multiple of the enzyme;

[0110] The results are shown in Table 4.

[0111] Table 4 Calculation results of enzymatic activity

[0112]

[0113] According to the detection method provided by the present application, the average enzyme activity calculated from three repeated reactions of two enzyme amount gradients is 8.06 U / μL, which is very close to the labeled activity of 8 U / ul of Bst DNA Polymerase (NEB), indicating that the method has good repeatability and high reliability.

[0114] Example 3 Detection of the activity of the enzyme to be tested

[0115] Step 1: Dilute the Bst DNA polymerase to be tested 5000 times, 10000 times, 20000 times, 30000 times, and 50000 times using enzyme diluent. This step needs to be prepared on ice and used immediately. Prepare the enzyme system to be tested on ice according to Table 3 (replace Bst DNA Polymerase in Table 3 with the enzyme to be tested), set up a negative control group without the enzyme to be tested, mix on a vortex shaker for 8 s, centrifuge in a microcentrifuge for 3 s, and make 3 replicate holes for each treatment (three repeats);

[0116] Step 2: The 5 groups of Bst DNA polymerase reaction systems and the negative control group obtained in the above configuration are placed in a fluorescence quantitative PCR instrument, and the fluorescence signal is monitored in real time (excitation / emission wavelength: 497 nm / 520 nm) under the condition of 65℃, 16s cycle for 40 times (constant temperature reaction); the fluorescence quantity change is shown in Table 6 (each dilution factor corresponds to 5×10 Figure 5 Figure 5 -3 -4 -4 -4 -4

[0117] Step 3: The data file is exported, the end-point fluorescence data of the last cycle of the Bst DNA polymerase reaction system to be tested in the data file is taken, the average fluorescence value of the 3 repeats of the negative control group is calculated, the end-point fluorescence value of each repeat of each gradient Bst DNA polymerase reaction system is subtracted by the average fluorescence value of the negative control, and the net fluorescence value of each repeat of each gradient Bst DNA polymerase reaction system is obtained. The average value of the net fluorescence value is calculated to obtain the average value of the net fluorescence value of the Bst DNA polymerase reaction system under each gradient (y1). The average value of the net fluorescence value is substituted into formula (A.1) to calculate the amount of newly generated double-stranded DNA (x1). The dNTP consumption amount y2 of each gradient is calculated according to formula (A.2), and the enzyme activity is calculated according to formula (A.3) by selecting the recommended dNTP consumption amount in the range of 0.0077 nmol-0.281 nmol, and the results are shown in Table 7.

[0118] Table 6 calculation results

[0119]

[0120] According to the method of the present application, the enzyme activity of the Bst DNA polymerase to be tested is calculated to be 157.963 U / μL.

[0121] The above is only a preferred embodiment of the present application, and does not limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the patent protection scope of the present application.​​​​​​​

Claims

1. A hairpin template for detecting Bst DNA polymerase activity, characterized in that, The nucleotide sequence of the hairpin template is shown in SEQ ID No.

1.

2. The use of the hairpin template for detecting Bst DNA polymerase activity as described in claim 1 in the preparation of a product for detecting Bst DNA polymerase activity.

3. A kit for detecting Bst DNA polymerase activity, characterized in that, Includes PCR reaction solution and DNA standard reaction solution; The PCR reaction solution includes an embedded fluorescent nucleic acid marker, a buffer solution, dNTPs, and a hairpin template for detecting Bst DNA polymerase activity as described in claim 1. The DNA standard reaction solution includes embedded fluorescent nucleic acid markers, buffer solution, and DNA standards.

4. The kit for detecting Bst DNA polymerase activity as described in claim 3, characterized in that, The embedded fluorescent nucleic acid label includes SYBR Green fluorescent nucleic acid dye or EvaGreen fluorescent nucleic acid dye; and / or, The dNTPs include dATP, dTTP, dCTP, and dGTP; and / or, The DNA standards include Lambda DNA standards.

5. The kit for detecting Bst DNA polymerase activity as described in claim 3, characterized in that, In the PCR reaction solution: The final concentration of the hairpin template used to detect Bst DNA polymerase activity is 0.25–0.4 μM; and / or, The dNTPs include dATP, dTTP, dCTP and dGTP, and the final concentrations of dATP, dTTP, dCTP and dGTP in the system are all 0.2 to 0.25 mM.

6. A method for detecting Bst DNA polymerase activity, characterized in that, Includes the following steps: S1. Provides the Bst DNA polymerase to be tested and the kit for detecting Bst DNA polymerase activity as described in any one of claims 3 to 5; S2. The DNA standard reaction solution is serially diluted to obtain DNA standard reaction solutions of multiple concentrations; the DNA standard reaction solutions of multiple concentrations are subjected to a first isothermal amplification treatment to obtain the fluorescence values ​​of the DNA standard reaction solutions of multiple concentrations; a standard curve formula is constructed by using the amount of DNA in the DNA standard reaction solutions of multiple concentrations and the corresponding fluorescence values. S3. Provide a Bst DNA polymerase to be tested, mix the Bst DNA polymerase to be tested with the PCR reaction solution and perform a second isothermal amplification treatment to obtain its endpoint fluorescence value; substitute the endpoint fluorescence value into the standard curve formula to obtain the amount of newly generated double-stranded DNA during the second isothermal amplification treatment. S4. The Bst DNA polymerase activity is calculated based on the amount of newly generated double-stranded DNA during the second isothermal amplification process.

7. The method for detecting Bst DNA polymerase activity as described in claim 6, characterized in that, In step S2, the conditions for the first isothermal amplification treatment are: constant temperature cycling at 60-65℃ for 90-100 times, with each cycle lasting 14-20 seconds.

8. The method for detecting Bst DNA polymerase activity as described in claim 6, characterized in that, In step S3, the conditions for the second isothermal amplification treatment are: constant temperature cycling at 60-65℃ for 30-50 cycles, with each cycle lasting 14-20 seconds.

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