A method and kit for detecting Bst DNA polymerase activity

Abstract

The application discloses a Bst DNA polymerase activity detection method and a kit thereof, and comprises the following steps: isothermal amplification is carried out by using Bst DNA polymerase, a standard curve of maximum fluorescence increment per unit time in a linear growth period of an amplification curve corresponding to double-stranded DNA of an amplification product of a Bst DNA polymerase standard is drawn, the maximum fluorescence increment per unit time in the linear growth period of an amplification product of a Bst DNA polymerase sample to be detected is detected, and the enzyme activity of the sample to be detected is calculated according to the standard curve. 2 The detection range of the enzyme activity is wider than 0.99, the detection accuracy is high, the repeatability is good, and the operation is simple when the enzyme activity is detected in the range of 10-80 U, and the Bst DNA polymerase activity can be quickly and accurately determined.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and more specifically, relates to a method and kit for detecting Bst DNA polymerase activity. Background Technology

[0002] Bacillus stearothermophilus (Bst) DNA polymerase is a member of the DNA polymerase A family. The full-length Bst DNA polymerase gene is 2634 bp, and the expressed protein is 878 amino acids long, corresponding to a molecular weight of 98 kDa. The larger fragment of Bst DNA polymerase, like Klenow's large fragment DNA polymerase, possesses 5′→3′ DNA polymerase activity, but lacks 5′→3′ exonuclease activity. It has lost 292 amino acids from the N-terminus, resulting in a gene length of 1758 bp, encoding a protein of 586 amino acids with a molecular weight of 67 kDa. Due to its strong thermostability, strand substitution activity, and polymerase activity, it is widely used in loop-mediated isothermal amplification (LAMP).

[0003] In the LAMP isothermal amplification method, Bst DNA polymerase uses double-stranded circular plasmid DNA as a template to achieve rapid and efficient amplification under isothermal conditions and with the help of amplification primers. It eliminates the need for template pre-denaturation, thus reducing the impact of temperature fluctuations in PCR technology and the requirement for expensive, sophisticated equipment. It offers advantages such as simple operation, high specificity, and easy product detection. Furthermore, Bst DNA polymerase can be used for sequencing GC-rich DNA sequences and for rapid sequencing of trace (nanogram) DNA templates. It plays a crucial role in various fields, including medical testing, pathogen inspection and quarantine, and food inspection. Therefore, accurately calibrating the activity of Bst DNA polymerase and ensuring the stability of enzyme activity across batches is of paramount importance.

[0004] Current methods for measuring Bst DNA polymerase activity typically employ isotope assays, meaning that the Bst DNA polymerase reaction system and conditions depend on the activity of radioactive elements. 3 The incorporation of H-labeled nucleotides synthesizes nucleotide chains labeled with radioactive isotopes, and the enzyme activity is then calculated by measuring the amount of radioactive isotope in the acid-insoluble product. However, this method is prone to radioactive contamination and requires multiple steps such as precipitation, washing, and drying, resulting in a long detection cycle, difficulty in achieving high throughput and automation, and posing challenges to the screening of Bst DNA polymerases.

[0005] Patent CN114561444A discloses a method for detecting Bst DNA polymerase activity. The principle is that the activity of Bst DNA polymerase is directly proportional to the amount of double-stranded DNA product within a certain range, and the amount of double-stranded DNA product is directly proportional to the amount of pyrophosphate in the reaction system. Therefore, the activity can be determined by the fluorescence intensity of a pyrophosphate reagent kit. While this method overcomes some of the shortcomings of the isotope method, it requires the addition of a pyrophosphate detection reagent after the isothermal amplification reaction, followed by the use of an ELISA reader to measure the corresponding signal for calculation. This process is relatively cumbersome and costly. Furthermore, the standard curve plotted by this method is an S-curve, not a non-linear positive correlation. That is, as enzyme activity increases, the pyrophosphate content initially increases and then tends to reach equilibrium. This indicates that the applicability of this detection method is limited, especially when detecting samples with high enzyme activity, where the detection accuracy is poor. Therefore, researching a widely applicable and highly accurate method for detecting Bst DNA polymerase activity is of great significance. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a method and kit for detecting Bst DNA polymerase activity. The aim is to discover that the maximum fluorescence increment per unit time of double-stranded DNA amplification products during the linear growth phase is linearly positively correlated with enzyme activity. By plotting a linear standard curve, Bst DNA polymerase activity can be rapidly determined. This solves the technical problems of existing Bst DNA polymerase activity detection methods, which use S-curves for standard curve plotting, resulting in a small detection range and poor accuracy in detecting high-enzyme-activity samples.

[0007] To achieve the above objectives, according to one aspect of the present invention, a method for detecting Bst DNA polymerase activity is provided, comprising the following steps:

[0008] Isothermal amplification was performed using Bst DNA polymerase. A standard curve was plotted showing the maximum fluorescence increment per unit time during the linear growth phase of the amplification curves corresponding to the Bst DNA polymerase standard and the amplified double-stranded DNA. The enzyme activity of the test sample was calculated based on the standard curve by detecting the maximum fluorescence increment per unit time of the amplified product of the Bst DNA polymerase during the linear growth phase.

[0009] Preferably, in the Bst DNA polymerase activity detection method, the unit time is the time required for one cycle of the isothermal amplification reaction.

[0010] Preferably, in the Bst DNA polymerase activity detection method, the isothermal amplification uses a set of isothermal amplification primers.

[0011] Preferably, in the Bst DNA polymerase activity detection method, the amplification primers include, but are not limited to, all conventional isothermal amplification primer sets, such as those shown in SEQ ID No. 1-6.

[0012] Preferably, in the Bst DNA polymerase activity detection method, the amplification primers have sequences as shown in SEQ ID No. 1-6.

[0013] Preferably, in the Bst DNA polymerase activity detection method, the isothermal amplification uses double-stranded circular plasmid DNA as the template, including but not limited to the product or template sequence of any primer set corresponding to any set of primers in the conventional isothermal amplification primer sets of the present invention, including the sequence shown in SEQ ID No. 7.

[0014] Preferably, in the Bst DNA polymerase activity detection method, the isothermal amplification uses a template double-stranded circular plasmid DNA as shown in SEQ ID No. 7.

[0015] Preferably, in the Bst DNA polymerase activity detection method, the maximum fluorescence increment is obtained by real-time quantitative amplification using a fluorescent dye method.

[0016] Preferably, the amplification reaction conditions of the Bst DNA polymerase activity detection method are 65°C, 15-60 seconds, 60 cycles; 85°C, 5 minutes.

[0017] According to another aspect of the present invention, a Bst DNA polymerase activity assay kit is also provided, comprising Bst DNA polymerase standards with an enzyme concentration of 5 to 80 U / μL.

[0018] In summary, compared with the prior art, the technical solutions conceived in this invention achieve the following beneficial effects because it was found that the maximum fluorescence increment per unit time of double-stranded DNA amplification products during the linear growth phase is linearly positively correlated with the enzyme amount:

[0019] The Bst DNA polymerase activity detection method provided by this invention rapidly determines the enzyme activity of a sample by plotting a standard curve showing the maximum fluorescence increment per unit time during the linear growth phase of the amplification curves corresponding to the Bst DNA polymerase standard and the double-stranded DNA amplification product. Because the plotted standard curve is linear, it has a wider range of applications, especially for detecting samples with enzyme activity in the range of 10–80 U, and offers high accuracy. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the principle of the enzyme activity detection method of the present invention;

[0021] Figure 2This is a standard curve of enzyme dosage versus maximum fluorescence value increment obtained by the detection method of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0023] Existing research indicates that Bst DNA polymerase activity can be rapidly determined by plotting a standard curve based on the pyrophosphate content of the isothermal amplified double-stranded DNA byproduct. However, the standard curve obtained by this method is an S-curve, where the pyrophosphate content initially increases and then tends to reach equilibrium as enzyme activity increases. In particular, the pyrophosphate content tends to reach equilibrium when the enzyme concentration reaches 16 U. This suggests that the detection range of this method is limited, especially when detecting samples with enzyme activity above 16 U / μL, where the accuracy of the detection results is poor.

[0024] This study measured the fluorescence intensity data of isothermal amplified double-stranded DNA using real-time quantitative PCR. By analyzing the changes in fluorescence data, an unexpected finding was that the maximum fluorescence increment per unit time during the linear growth phase of the amplification curve was positively correlated with Bst DNA polymerase activity. Further analysis of the maximum fluorescence increment per unit time during the linear growth phase for different concentrations of Bst DNA polymerase standards revealed a linear positive correlation in the standard curve, expressed as y = 0.3254x - 0.0113, where x represents Bst DNA polymerase activity, y represents the maximum fluorescence increment per unit time during the linear growth phase, and R0... 2 Above 0.99, the Bst DNA polymerase activity of the test sample can be calculated by measuring the maximum fluorescence increment per unit time during the linear growth phase of the double-stranded DNA amplification product. This method has a wider detection range and higher accuracy.

[0025] The present invention provides a method for detecting Bst DNA polymerase activity, which specifically includes the following steps:

[0026] (1) Plot the maximum fluorescence increment-enzyme activity standard curve: Using double-stranded circular plasmid DNA as a template, in the presence of dNTP mix and a set of isothermal amplification primers, Bst DNA polymerase is used for isothermal amplification to obtain the maximum fluorescence increment per unit time in the linear growth phase of the amplification curve corresponding to multiple standards with different enzyme activity concentrations, and plot the maximum fluorescence increment-enzyme activity standard curve.

[0027] The standard curve is a linear positive correlation; for example, in some embodiments, the standard curve plotted is y = 0.3254x - 0.0113, R0 2 =0.9982, where x is the activity of Bst DNA polymerase and y is the maximum fluorescence increment per unit time during the linear growth phase of the amplification curve;

[0028] Compared to the standard curve obtained by the detection method in patent CN114561444A, which shows an initial increase in pyrophosphate content followed by a tendency to reach equilibrium with increasing enzyme activity, and reaches equilibrium at an enzyme concentration of 16 U, the accuracy of the detection results is poor when the enzyme activity is above 16 U / μL. In contrast, the standard curve provided by this invention exhibits a linear positive correlation, a wider detection range, and higher accuracy when detecting samples with enzyme activity above 16 U / μL.

[0029] (2) Determine the activity of Bst DNA polymerase: Isothermal amplification is used to obtain the maximum fluorescence increment per unit time during the linear growth period of the amplification curve of the enzyme sample to be tested. Substitute this into the standard curve obtained in step (1) to calculate the activity of the enzyme sample to be tested, which is the determined Bst DNA polymerase activity.

[0030] Furthermore, in the method for detecting Bst DNA polymerase activity, the maximum fluorescence increment is obtained by measuring with a real-time quantitative PCR instrument; the unit time is the time required for one cycle of the isothermal amplification reaction (LAMP reaction), and in some embodiments, the unit time is 30s.

[0031] In some embodiments, the maximum fluorescence increment per unit time during the linear growth period of the amplification curve is obtained by real-time quantitative fluorescence amplification using a fluorescent dye method.

[0032] The isothermal amplification is performed using double-stranded circular plasmid DNA as a template, under the conditions of dNTP mix and a set of isothermal amplification primers; the isothermal amplification primers include, but are not limited to, all conventional LAMP primer sets, including the sequences shown in SEQ ID No. 1-6.

[0033] The double-stranded circular plasmid DNA includes, but is not limited to, all products or template sequences corresponding to any set of conventional LAMP primers in this invention, including the sequence shown in SEQ ID No. 7.

[0034] The amplification reaction procedure is set according to the selected double-stranded circular plasmid DNA template and amplification primers to achieve effective amplification.

[0035] In some embodiments of the amplification reaction procedure, the template is a double-stranded circular plasmid DNA as shown in SEQ ID No. 7. The amplification reaction conditions are: 65℃, 15-60 seconds, 60 cycles; 85℃, 5 minutes; fluorescence channel: SYBR, with automatic collection of fluorescence signal at the end of each cycle. The maximum fluorescence increment is obtained using a real-time quantitative PCR instrument, as detailed below:

[0036] After real-time isothermal amplification is complete, first terminate the Bst DNA polymerase amplification reaction, export the obtained fluorescence value data, and refer to the fluorescence value range and ct value range of its amplification curve during the linear growth phase, as shown below. Figure 1 As shown, the maximum fluorescence value increment per unit time within the exported data is then found, which is the maximum fluorescence increment per unit time during the linear growth phase of the amplification curve.

[0037] In addition, the present invention also provides a Bst DNA polymerase activity assay kit, which includes Bst DNA polymerase standards with an enzyme concentration of 5 to 80 U / μL, preferably including at least two Bst DNA polymerase standards with an enzyme concentration of 5 to 80 U / μL and at least one set of LAMP primers.

[0038] The following is an example:

[0039] Example 1: Determination of Bst 2.0 DNA Polymerase (NEB) Activity

[0040] (1) Plotting the standard curve

[0041] This embodiment uses double-stranded circular plasmid DNA as a template. In the presence of dNTP mix and a set of isothermal amplification primers, Bst DNA polymerase is used for isothermal amplification. The amplification curve of the double-stranded DNA product is obtained using a real-time quantitative PCR instrument. A standard curve is plotted based on the maximum fluorescence increment per unit time during the linear growth phase of the amplification curve using Bst DNA polymerase standards. The x-axis represents Bst DNA polymerase activity, and the y-axis represents the maximum fluorescence increment. The activity of the Bst DNA polymerase to be tested can be calculated from the standard curve. The standard curve is plotted by subtracting the net fluorescence increment at x=0 from the fluorescence increment at each concentration. The specific operation is as follows:

[0042] ① Reagents required for isothermal amplification reaction: as shown in Table 1.

[0043] Table 1. Reagents required for isothermal amplification reaction

[0044]

[0045] ② Operational steps of the isothermal amplification reaction

[0046] First, prepare the reaction mixture according to Table 2. Five enzyme concentration gradients of Bst Plus DNA Polymerase (40 U / μL) were set up: 80 U, 40 U, ​​20 U, 10 U, and 0 U; that is, 2 μL, 1 μL, 0.5 μL, 0.25 μL, and 0 μL were added to the system, respectively. After mixing and centrifugation, the reaction mixtures containing each enzyme concentration gradient were placed on a real-time quantitative PCR instrument for reaction. The reaction was carried out at 65℃ for 30 s for 60 cycles, followed by 85℃ for 5 min. The fluorescence channel was SYBR, and the fluorescence signal was automatically collected at the end of each cycle.

[0047] Table 2 Reference System for Isothermal Amplification Reaction Mixture

[0048]

[0049]

[0050] Note: 10×primers:(FIP:BIP:F3:B3:LF:LB) is

[0051] 12μM:12μM:2μM:2μM:8μM:8μM.

[0052] ③ Draw the standard curve

[0053] After the reaction, the fluorescence values ​​of each enzyme concentration were exported, and the fluorescence value range and ct value range of their amplification curves during the linear growth phase were referenced. Figure 1 As shown, the maximum fluorescence value increment within a unit time of 30 seconds was found in the exported data. Using enzyme dosage (U) as the x-axis and maximum fluorescence value increment (RFL) as the y-axis, a standard curve for the commercial enzyme was plotted using OriginPro 8.6 with a non-linear regression curve. It was found that these data points could fit a standard curve very well, as shown... Figure 2 As shown, and its R 2 A value >0.99 indicates a linear positive correlation between the maximum fluorescence value increment and enzyme activity. The enzyme activity of the test sample can be calculated by obtaining the maximum fluorescence value increment per unit time during the linear growth period.

[0054] The specific maximum fluorescence value increments corresponding to different enzyme activities are shown in Table 3 below.

[0055] Table 3. Different enzyme concentration gradients and corresponding fluorescence values

[0056] Serial Number Enzyme dosage in the system (U) Maximum fluorescence value increment 1 0 0 2 10 0.31024 3 20 0.65698 4 40 0.94158 5 80 1.29996

[0057] (2) Determination of Bst 2.0 DNA Polymerase (NEB) activity

[0058] ①Isothermal amplification reaction: The reagents required for the isothermal amplification reaction are shown in Table 4.

[0059] Table 4. Reagents for Isothermal Amplification Reaction

[0060]

[0061]

[0062] Note: Bst 2.0 DNA Polymerase (NEB), commercially available enzyme activity is 8 U / μL, its enzyme activity is defined as (30 min, consumption of 25 mmol of dNTPs), Yeasen brand. Bst Plus DNA Polymerase (40 U / μL), enzyme activity defined (30 min, 10 mmol dNTP consumption), was converted to the enzyme activity of the test sample Bst 2.0 DNA Polymerase (NEB) at the enzyme activity definition (30 min, 10 mmol dNTP consumption) of 20 U / μL.

[0063] ②Isothermal amplification reaction operation steps

[0064] The assay of the test samples and the standard curve experiment were performed simultaneously, using the same reaction system and operating procedures. The test samples were Bst 2.0 DNA Polymerase (NEB), with a commercially available enzyme labeling activity of 20 U / μL. Isothermal amplification was performed. The preparation of the isothermal amplification reaction mixture is shown in Table 2. The enzyme volume for the test samples was 1 μL, and three parallel experiments were conducted.

[0065] Reaction conditions: 65℃ for 30s, 60 cycles; 85℃ for 5min; Fluorescence channel: SYBR, fluorescence signal automatically collected at the end of each cycle.

[0066] ③ Data processing

[0067] Export the fluorescence data for each enzyme concentration gradient, and refer to the fluorescence value range and ct value range of its amplification curve during the linear growth phase. Then, find the maximum fluorescence value increment per unit time in the exported data, substitute it into the standard curve for calculation, and obtain the relative enzyme activity of the sample to be tested. The specific results are shown in Table 5.

[0068] Table 5. Results of Bst 2.0 DNA Polymerase (NEB) enzyme activity assay in the samples to be tested.

[0069]

[0070] As shown in Table 5, the calculated enzyme activity of 20.816 U / μL obtained by the determination method provided in this invention is highly consistent with the actual enzyme activity of Bst2.0 DNA Polymerase (NEB) of 20 U / μL (30 min, consuming 10 mmol of dNTPs). Moreover, the CV% of the same sample was <5% after three repeated tests, indicating that the reproducibility of this method is good and its reliability is high.

[0071] Example 2: Determination of Bst 2.0HS (BiORi) activity

[0072] (1) Plotting the standard curve: using A standard curve was prepared using Bst Plus DNA Polymerase (40 U / μL) (Yeasen: 14402ES92), following the same steps as in Example 1.

[0073] (2) Determination of Bst 2.0HS (BiORi) activity

[0074] ①Isothermal amplification reaction: The reagents required for the isothermal amplification reaction are shown in Table 6.

[0075] Table 6. Reagents for Isothermal Amplification Reaction

[0076]

[0077]

[0078] ②Isothermal amplification reaction operation steps

[0079] The assay of the test sample and the standard curve experiment were performed simultaneously, using the same reaction system and operating procedures. The test sample was Bst 2.0HS (BiORi), with a commercially available enzyme labeling activity of 8 U / μL. An isothermal amplification reaction was conducted. The preparation of the isothermal amplification reaction mixture is shown in Table 2. The enzyme volume for the test sample was 1 μL, and three parallel experiments were performed.

[0080] Reaction conditions: 65℃ for 30s, 60 cycles; 85℃ for 5min; Fluorescence channel: SYBR, fluorescence signal automatically collected at the end of each cycle.

[0081] ③ Data processing

[0082] Export the fluorescence values ​​for each enzyme concentration gradient, and refer to the fluorescence value range and ct value range of their amplification curves during the linear growth phase. Figure 1 Then, find the maximum fluorescence value increment per unit time in the exported data, substitute it into the standard curve for calculation, and obtain the relative enzyme activity of the sample to be tested. The measurement results are shown in Table 7.

[0083] Table 7 Results of Bst 2.0HS (BiORi) enzyme activity assay in the samples to be tested

[0084]

[0085]

[0086] As shown in Table 7, the calculated enzyme activity of 8.3378 U / μL obtained by the determination method provided by the present invention is highly consistent with the actual enzyme activity of Bst 2.0HS (BiORi) of 8 U / μL (30 min, consuming 10 mmol of dNTP). Moreover, when testing samples with low enzyme activity, the same sample was tested three times, and the CV% was <5%, indicating that the repeatability of this method is good and the reliability is high.

[0087] Example 3: Determination of Bst DNA Polymerase (NanoMagBio) Activity

[0088] (1) Plotting the standard curve: using A standard curve was prepared using Bst Plus DNA Polymerase (40 U / μL) (Yeasen: 14402ES92), following the same steps as in Example 1.

[0089] (2) Determination of Bst DNA Polymerase (NanoMagBio) activity

[0090] ① Isothermal Amplification Reaction Procedure: The reagents required for the isothermal amplification reaction are shown in Table 1 of Example 1. The sample determination and standard curve experiment were performed simultaneously, with the same reaction system and operating procedures. The sample to be tested was our company's own Bst DNA Polymerase. The Bst DNA Polymerase was serially diluted twofold using enzyme dilution buffer to obtain enzyme concentration gradients of 2×, 4×, 8×, 16×, 32×, 64×, and 128×. Then, the isothermal amplification reaction was performed. The preparation of the isothermal amplification reaction mixture is shown in Table 2 of Example 1. The enzyme volume for each sample was 1 μL, and three parallel experiments were conducted for each concentration gradient.

[0091] Reaction conditions: 65℃ for 30s, 60 cycles; 85℃ for 5min; Fluorescence channel: SYBR, fluorescence signal automatically collected at the end of each cycle.

[0092] ③ Data processing

[0093] First, based on the amplification results, the maximum enzyme dilution concentration gradient that can be amplified was determined. Then, the fluorescence value data of the two enzyme dosage gradients adjacent to the maximum amplification gradient were exported. Referring to the fluorescence value range and ct value range of their amplification curves during the linear growth phase, the maximum fluorescence value increment per unit time within this range was found in the exported data and substituted into the standard curve for calculation to obtain the relative enzyme activity of the sample. In this experiment, the maximum enzyme dilution concentration gradient that could be successfully amplified was 64×. Therefore, the fluorescence value data of the adjacent enzyme dilution concentration gradients of 16×, 32×, and 64× were exported and substituted into the standard curve for calculation. The results calculated according to the standard curve showed high consistency with the actual enzyme activity. Taking the maximum enzyme dilution concentration gradient of 64× as an example, the specific measurement results are shown in Table 8.

[0094] Table 8 Results of Bst DNA Polymerase (NanoMagBio) Enzyme Activity Detection in Samples

[0095]

[0096] As shown in Table 8, the calculated enzyme activity of 20.412 U / μL obtained by the determination method provided in this invention is highly consistent with the actual enzyme activity of BstDNA Polymerase (NanoMagBio) of 20 U / μL. Moreover, the CV% of the same sample was less than 5% after three repeated tests, indicating that the reproducibility of this method is good and its reliability is high.

[0097] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting Bst DNA polymerase activity, characterized in that, Includes the following steps: Using double-stranded circular plasmid DNA as a template, dNTP mix and a set of isothermal amplification primers were added. Isothermal amplification was performed using Bst DNA polymerase standard. The amplification curve of the double-stranded DNA product was obtained by real-time fluorescence quantitative PCR. The maximum fluorescence increment per unit time during the linear growth phase of the amplification curves corresponding to multiple standards with different enzyme activity concentrations was obtained. A standard curve was plotted showing the maximum fluorescence increment per unit time during the linear growth phase of the amplification curves corresponding to the enzyme amount of Bst DNA polymerase standard and the amplification product double-stranded DNA. The unit time is the time required for one cycle of the isothermal amplification reaction, and the standard curve shows a linear positive correlation. The enzyme activity of the test sample was calculated based on the standard curve by detecting the maximum fluorescence increment per unit time of the amplification product of Bst DNA polymerase during the linear growth phase.

2. The method for detecting Bst DNA polymerase activity as described in claim 1, characterized in that, The amplification primers include sequences as shown in SEQ ID No. 1-6.

3. The method for detecting Bst DNA polymerase activity as described in claim 2, characterized in that, The sequence of the set of isothermal amplification primers is shown in SEQ ID No. 1-6.

4. The method for detecting Bst DNA polymerase activity as described in claim 3, characterized in that, The isothermal amplification uses a template double-stranded circular plasmid DNA as shown in SEQ ID No.

7.

5. The method for detecting Bst DNA polymerase activity as described in claim 1, characterized in that, The maximum fluorescence increment was obtained using real-time quantitative fluorescence amplification with fluorescent dyes.

6. The method for detecting Bst DNA polymerase activity as described in claim 5, characterized in that, The amplification reaction conditions were 65℃ for 15-60 seconds for 60 cycles, and 85℃ for 5 minutes.

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