Absolute quantification method of antibody taq dna polymerase activity based on real-time fluorescence detection

Abstract

The present application relates to the technical field of molecular biology detection, in particular to an absolute antibody Taq DNA polymerase activity determination method based on real-time fluorescence quantitative detection, comprising the following steps: firstly, establishing a standard curve of DNA content and fluorescence value by reacting different concentrations of double-stranded lambda DNA with fluorescent dyes; then, performing an extension reaction in a specific reaction system using M13 single-stranded DNA as a template, recording fluorescence values in real time, calculating net fluorescence values and establishing a linear relationship between the net fluorescence values and reaction time; converting the amount of newly generated double-stranded DNA at different time points by using the standard curve, further calculating the consumption amount of dNTPs, and finally obtaining the absolute activity of the antibody Taq DNA polymerase according to the definition of the activity of the antibody Taq DNA polymerase. The whole process combines fluorescence detection and a standard curve to realize accurate determination of enzyme activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology detection technology, and particularly to an absolute value method for antibody Taq DNA polymerase activity based on real-time fluorescence quantitative detection. BACKGROUND

[0002] DNA polymerase is a key enzyme in molecular biology, which catalyzes the polymerization of deoxy-ribonucleoside triphosphate (dNTP) molecules to form a progeny DNA with the parent DNA as a template. The DNA polymerase I was first discovered in Escherichia coli by American scientist Arthur Komberg in 1957. The Klenow fragment of DNA polymerase I was used in the early stage of the Polymerase Chain Reaction (PCR) technology. Since it is not heat-resistant and will be inactivated at high temperatures, new polymerase needs to be supplemented in each cycle, which is very troublesome and limits the development of PCR technology.

[0003] In 1988, Saiki et al. successfully applied a more stable heat-resistant DNA polymerase isolated from Thermus aquaticus to PCR technology, which greatly improved the efficiency of DNA amplification. This polymerase is the well-known Taq DNA polymerase. After that, the PCR method has been continuously improved, and the DNA polymerase used has also been continuously optimized, resulting in more types. Currently, there are a variety of commercial products, each with its own characteristics, mainly including Taq series, Pfu series, Vent series, KOD series, T7, Tth, etc.

[0004] DNA polymerase is widely used in the biopharmaceutical industry, including genetic engineering product production, process optimization, new drug research and development, medical diagnosis and monitoring, and basic research in life sciences. In genetic testing and analysis, DNA polymerase can be used for diagnosing genetic diseases, performing DNA fingerprinting, etc. by amplifying and analyzing specific genes or DNA sequences; in drug research and development, it can be used to evaluate drug toxicity, screen candidate drugs, study drug metabolism and targets, etc.; in environmental microbiology and population genetics research, it can amplify and analyze the DNA of various biological systems; in medical diagnosis, the high sensitivity and specificity of PCR technology combined with DNA polymerase can be used for early disease diagnosis, detection of infectious diseases, genetic diseases, and cancer markers, etc.

[0005] The activity of DNA polymerase is generally defined by reference methods, and the same enzyme can have different activity values according to different activity definitions. Therefore, establishing the source of enzyme activity value and researching the establishment of enzyme activity quantitative method are the main contents of quality control. At present, the commonly used activity determination methods of DNA polymerase mainly include the following:

[0006] 1. Isotope labeling method (radioactive incorporation method): This method has extremely high sensitivity and can directly reflect the enzyme reaction rate, but it has radioactive hazards, special protection is required for operation, the waste treatment steps are complicated, and the half-life of the isotope limits the storage time of the reagent;

[0007] 2. Colorimetric method (pyrophosphate release method): simple operation, no need for complex equipment, low cost, but low sensitivity, easy to be disturbed by impurities (such as free phosphate), and the experimental conditions need to be strictly optimized;

[0008] 3. Real-time quantitative PCR method (qPCR method): high sensitivity and accurate quantitative characteristics, close to the actual application scene (PCR), but specific primers / probes are required and the reaction system (such as template purity, primer design, etc.) needs to be strictly standardized;

[0009] 4. National standard method (GB / T 35542-2017): Although this method also uses fluorescence quantitative technology, the template / primers select a shorter palindromic sequence, which limits the upper limit of fluorescence collection, is easy to cause deviation, and is not easy to collect the linear amplification interval, which puts higher requirements on the experimenters.

[0010] Therefore, a safe, convenient, accurate and reproducible DNA polymerase activity determination method is urgently needed to meet the quality control requirements of DNA polymerase in the biological and pharmaceutical industries. SUMMARY

[0011] In view of the problems in the prior art, the purpose of the present application is to provide an antibody Taq DNA polymerase activity absolute determination method based on real-time fluorescence quantitative detection, which is safe, convenient, rapid, sensitive, accurate and reproducible, solves the problem of radioactive hazards in the isotope labeling method in the prior art, overcomes the low sensitivity of the colorimetric method, and improves the problem of upper limit of fluorescence collection caused by improper selection of templates in the national standard method.

[0012] To achieve the above purpose, the present application provides an antibody Taq DNA polymerase activity absolute determination method based on real-time fluorescence quantitative detection, comprising the following steps:

[0013] a) Using different concentrations of double-stranded lambda DNA as standard, adding saturated nucleic acid fluorescent dye, recording the fluorescence value, and constructing a standard curve of double-stranded lambda DNA content and fluorescence value;

[0014] b) using M13 phage single-stranded DNA as a template, adding the antibody Taq DNA polymerase to be tested into a reaction system containing M13R primer, dNTPs and saturated nucleic acid fluorescent dye, and initiating an extension reaction, and recording the fluorescence values at different time points in real time under the condition of 74°C;

[0015] c) calculating the net fluorescence values at different time points during the reaction, and establishing a linear relationship between the net fluorescence values and the reaction time;

[0016] d) calculating the amount of newly generated double-stranded DNA at different time points in step c) according to the standard curve established in step a);

[0017] e) calculating the dNTP consumption according to the amount of newly generated double-stranded DNA;

[0018] f) calculating the absolute activity of the antibody Taq DNA polymerase to be tested according to the definition of antibody Taq DNA polymerase activity.

[0019] Preferably, the concentration gradient of the double-stranded λ DNA standard in step a) is 0, 5, 10, 25, 50, 100 ng.

[0020] Preferably, the saturated nucleic acid fluorescent dye is EvaGreen fluorescent dye, and the final concentration is 0.8×.

[0021] Preferably, the reaction system in step b) further comprises: 1× activity measurement Buffer; 3mM MgCl2; 0.2mM dNTPs; 0.4μM M13R primer; 200ng / μL M13 mp18 single-stranded DNA.

[0022] Preferably, the sequence of the M13R primer in step b) is 5'-GTTGTAAAACGACGGCCAG-3'.

[0023] Preferably, the concentration of the antibody Taq DNA polymerase to be tested in step b) is 0.008U / μL.

[0024] Preferably, the calculation formula of the dNTP consumption in step e) is: dNTP consumption (nmol) = newly generated double-stranded DNA amount (ng) / (2×324.5), wherein 2 represents that double-stranded DNA is composed of two single-stranded DNAs, and 324.5 is the relative average molecular weight of dNTP.

[0025] Preferably, the calculation formula of the activity of the antibody Taq DNA polymerase to be tested in step f) is: enzyme amount (U) = newly generated double-stranded DNA amount (ng) × 3 / (2×324.5×10) = newly generated double-stranded DNA amount (ng) × 4.62×10⁻ 4Wherein, the amount of newly generated double-stranded DNA is the amount generated in 10 minutes, multiplied by 3 to represent the amount generated in 30 minutes, and divided by 10 to represent the number of times the amount of dNTP consumed in 30 minutes is 10 nmol.

[0026] Preferably, the calculation formula of the absolute activity of the mother liquor in step f) is: absolute activity of the mother liquor (U / μL) = (amount of enzyme / 5) x total dilution factor, wherein 5 represents the amount of enzyme added in the final reaction system, and the total dilution factor is the dilution factor from the enzyme mother liquor to the final reaction system.

[0027] Preferably, the PCR reaction program in step b) comprises: performing 3 readings of fluorescence values at 74°C as initial values; 95°C, 1 minute activation; 74°C, 30 seconds, 30 cycles, and collecting fluorescence signals once per cycle.

[0028] The beneficial effects of the present application include:

[0029] 1. Safe and convenient: The present application uses fluorescent dye detection technology, which avoids the radioactivity hazards of isotope labeling method, does not require special protection and cumbersome waste treatment, and is safer and more convenient to operate.

[0030] 2. Fast and sensitive: Through real-time fluorescent quantitative PCR technology, the DNA polymerization process can be monitored in real time, with high sensitivity and fast detection speed.

[0031] 3. Accurate measurement: The present application optimizes the template selection, uses M13 filamentous bacteriophage single-stranded DNA as the template, and overcomes the upper limit restriction of fluorescence collection caused by the short palindromic sequence in the national standard method, making the measurement more accurate. Experimental verification shows that the CV values of the activity measurement results of five enzymes in the same batch are all less than 5%, indicating high measurement accuracy.

[0032] 4. Good repeatability: By optimizing the reaction conditions and calculation method, a stable double-standard curve system is established, realizing accurate conversion from fluorescence signal to enzyme activity, and ensuring the repeatability of the measurement results. Statistical analysis shows that the measurement results conform to the normal distribution and have no abnormal values, proving that the method is reliable and stable.

[0033] 5. Low cost: Compared with the isotope labeling method, the present method does not require the use of expensive radioactive isotopes, reducing the measurement cost.

[0034] 6. Suitable for high-throughput detection: The present method is simple to operate and can handle multiple samples simultaneously, suitable for high-throughput DNA polymerase activity detection requirements. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Linear relationship diagram of fluorescence values and cycle numbers of Taq DNA polymerase with different concentrations of antibodies.

[0036] Figure 2 Figure 1 is a graph of the change in the fluorescence value of the antibody Taq DNA polymerase over time.

[0037] Figure 3 Figure 2 is a standard curve graph constructed from the dsDNA standard (ng) and ΔRn.

[0038] Figure 4 Figures 3-7 are graphs of the relationship between the fluorescence signal value and time during the reaction of test enzymes 1-5, respectively (each repeated 3 times).

[0039] Figure 5 Figures 3-7 are graphs of the relationship between the fluorescence signal value and time during the reaction of test enzymes 1-5, respectively (each repeated 3 times).

[0040] Figure 6 Figures 3-7 are graphs of the relationship between the fluorescence signal value and time during the reaction of test enzymes 1-5, respectively (each repeated 3 times).

[0041] Figure 7 Figures 3-7 are graphs of the relationship between the fluorescence signal value and time during the reaction of test enzymes 1-5, respectively (each repeated 3 times).

[0042] Figure 8 Figures 3-7 are graphs of the relationship between the fluorescence signal value and time during the reaction of test enzymes 1-5, respectively (each repeated 3 times).

[0043] Figure 9 Figure 8 is a Q-Q graph of the antibody Taq DNA polymerase activity data of the five groups. DETAILED DESCRIPTION

[0044] The application will be further described in conjunction with specific examples, but the scope of the application is not limited thereto.

[0045] Example 1: The application provides an absolute value method for the activity of antibody Taq DNA polymerase based on real-time fluorescence quantitative detection. The method uses the property of EVAGreen fluorescent dye that specifically binds to double-stranded DNA, and establishes a standard curve by the amount of double-stranded λ DNA standard DNA and the fluorescence value. At the same time, single-stranded DNA of M13 filamentous bacteriophage is used as a template to extend in a reaction system containing enzyme, primer, fluorescent dye and dNTPs, etc., and the net fluorescence value of newly generated double-stranded DNA at different time points is recorded. In the early stage of the reaction, there is a linear relationship between time and net fluorescence value, and a linear equation between the two is fitted, and the consumption of dNTPs at different time points is calculated by combining the standard curve. The activity of the DNA polymerase to be tested is calculated according to the definition of the enzyme activity of DNA polymerase.

[0046] I. Reagents and instruments:

[0047] The main instruments and reagents required for the method of the present application include:

[0048] 1.1 PCR instrument: ABI QuantStudio 12K full-featured fluorescent quantitative PCR instrument.

[0049] 1.2 Pipette gun: specifications 10 uL, 20 uL, 100 uL, 200 uL, 1000 uL.

[0050] 1.3 Reagents: sterilized water, antibody Taq DNA polymerase activity standard substance, EvaGreen™ saturated fluorescent dye, 10uM M13R primer (5 ， -GTTGTAAAACGACGGCCAG-3 ， ), 25mM MgCl2, 300ng / uL dsDNA, 2.5mM dNTP, 200ng / uL M13 mp18 Single Strand DNA, 10x activity measurement Buffer.

[0051] II. Determination of antibody Taq DNA polymerase activity standard experiment conditions

[0052] 2.1 Activity measurement Buffer (1x)

[0053] Take an appropriate volume of activity measurement Buffer (10x), dilute ten times with sterilized water to obtain activity measurement Buffer (1x), mix well and reserve.

[0054] 2.2 Mix1 solution

[0055] Prepare Mix1 solution according to Table 1 by adding the corresponding volume of solution, mix well, centrifuge, and store at 4°C for use within 12 hours.

[0056] Table 1 Mix1 composition

[0057]

[0058] 2.3 Double-stranded reaction system Mix2 solution

[0059] Prepare double-stranded reaction system Mix2 solution (20uL / portion), take Mix1 solution 345uL (15uL / tube x 23 tubes), add sterilized water 115uL (5uL / tube x 23 tubes), mix well, centrifuge and reserve.

[0060] 2.4 Single-stranded reaction system Mix3 solution

[0061] Prepare single-stranded reaction system Mix3 solution (20ul / portion), see Table 2

[0062] Table 2 Composition of single strand reaction system Mix3

[0063] 2.5 Gradient concentration double-stranded dsDNA solution

[0064] Dilute the double-stranded dsDNA solution with the Activity Measurement Buffer (1x), as shown in Table 3.

[0065] Table 3 Diluted double-stranded dsDNA

[0066]

[0067] 2.6 Dilution of enzyme to be tested

[0068] First estimate the approximate activity of the enzyme to be tested, dilute it to 0.008 U / uL for activity determination. The estimated activity of the antibody Taq DNA polymerase is about 5 U / uL. First dilute the enzyme to be tested to 1 U / uL (minimum sampling volume 5 uL) with 1x Activity Measurement Buffer, then dilute the 1 U / uL enzyme to be tested to 0.08 U / uL, then dilute the 0.08 U / uL sample to 0.008 U / uL, as shown in Table 4. In the previous condition optimization, it was verified through experiments that the antibody Taq DNA polymerase at 0.005, 0.008, 0.0125 U / uL had a linear relationship between fluorescence value and time in the determination range (R2> 0.99) Figure 1 Therefore, 0.008 U / uL was selected as the final concentration for enzyme activity determination, and a reaction system with the addition of equal volume of 1x Activity Measurement Buffer was used as a negative control.

[0069] Table 4 Dilution of enzyme to be tested

[0070]

[0071] 2.7 Reaction system dispensing

[0072] 3.2.7.1 Double-stranded system: Mix2 20ul + dsDNA (40, 20, 10, 5, 2, 1, 0 ng / ul) 5ul;

[0073] 3.2.7.2 Single-stranded system: Mix3 20ul + enzyme to be tested / 1x Activity Measurement Buffer 5ul;

[0074] In the method validation, five tubes of antibody Taq DNA polymerase of the same batch were selected for determination, three parallel test holes were arranged for each test point, and the reaction system with the addition of equal volume of 1x activation buffer was used as negative control, a total of 18 test holes, centrifuged and detected as soon as possible. The double-stranded system and the single-stranded system were added to the 96-well plate of the ABI QuantStudio12K full-function fluorescence quantitative PCR instrument and sealed. Note: The above experimental operations are carried out on ice.

[0075] 2.8 Fluorescence quantitative PCR program setting

[0076] The double-stranded standard curve does not need to be activated, and the fluorescence value is read and recorded for 3 times at 74°C first, then the reaction conditions are set according to the operation rules of the PCR instrument, 95°C, 1 min activation, and the reaction conditions are set again (74°C, 30s) for 30 cycles, the reaction system is set to 25ul, the fluorescence is selected SYBR Green, the signal is collected once for each cycle, the reaction is started, and the fluorescence signal change in the whole reaction process is detected. Figure 2 ).

[0077] Three, antibody Taq DNA polymerase activity determination data processing

[0078] 3.1 The fluorescence signal value of the output reaction is saved to the excel table.

[0079] 3.2 Making standard curve

[0080] The average value and standard error of the fluorescence signal of each double-stranded gradient are calculated, and the average value of the fluorescence signal of each gradient is subtracted from the average value of the fluorescence signal of the background 0ng dsDNA to obtain ∆Rn. The amount (ng) of the standard (dsDNA) is taken as the abscissa, and ∆Rn is taken as the ordinate. A binomial fitting curve is made. In the formula of the binomial curve, all numbers are kept to 5 decimal places. The correlation coefficient R 2 is kept to 4 decimal places, and R 2 should be greater than 0.9900. The fluorescence value data of the standard curve is shown in Table 5, the relationship curve of ∆Rn and the amount (ng) of the standard (dsDNA) is shown in Figure 3 , and the standard curve equation is obtained by fitting: y=36411.13936 × x +6000.38585, R 2 =0.9934.

[0081] Table 5 Standard curve fluorescence value raw data

[0082]

[0083] 3.3 Making fluorescence value and time correlation curve

[0084] A binomial curve was plotted with the time of each fluorescence recording point in each enzyme experimental group as the x-axis and the fluorescence value Rn of each well as the y-axis. All numbers in the binomial curve formula were rounded to five decimal places. The correlation coefficient R... 2 Rounded to 4 decimal places, R 2 It should be greater than 0.9900. The fluorescence signal values ​​at each time point for the three enzyme tubes tested (numbered 1, 2, 3, 4, 5) are shown in Tables 6-1 and 6-2. The relationship curve between Rn and time is shown in... Figures 4-8 As shown, the fitting yields the calculation equation, enzyme 1-1: y = 3950.38970 × x + 1513625.93040 (R²) 2 =0.9994), enzyme 1-2: y=3850.78232 × x +1296205.11065 (R 2 =0.9995), enzyme 1-3: y=4042.27612 × x + 1377814.11069 (R 2 =0.9996); Enzyme 2-1: y = 4165.48428 × x + 1597069.61703 (R 2 =0.9996), enzyme 2-2: y=3821.48545 × x +1420394.96913 (R 2 =0.9997), enzyme 2-3: y=3861.44174 × x + 1489822.37290 (R 2 =0.9997); Enzyme 3-1: y = 3915.99742 × x + 1570762.81000 (R 2 =0.9998), enzyme 3-2: y=4142.79985 × x +1773173.84616 (R 2 =0.9998), enzyme 3-3: y=3891.92021 × x + 1571821.88319 (R 2 =0.9998); Enzyme 4-1: y=4171.31808 × x + 1720903.70500, Enzyme 4-2: y=4164.29177 × x +1482471.62507, Enzyme 4-3: y=4256.02596 × x + 1523817.60627; Enzyme 5-1: y= 4130.29271 × x + 1646131.15877, Enzyme 5-2: y=4125.19525 × x + 1508695.84083, Enzyme 5-3: y=4443.56832 × x + 1650168.23246.

[0085] Table 6-1 Raw data of fluorescence signal value during the test reaction process

[0086]

[0087] Table 6-2 Raw data of fluorescence signal value during the test reaction process

[0088]

[0089] 3.4 Calculation of dsDNA production (ng)

[0090] Taking the extension time of 10 min (e.g. 150s-750s, a total of 600s) into the formulas fitted in 3.3, the increment of fluorescence value ΔRn can be calculated, and the standard curve in 3.2 can be calculated to calculate the double-stranded DNA production (ng) generated in 10 min.

[0091] 3.5 Calculation of dNTP consumption (nmol)

[0092] dNTP consumption (nmol) = dsDNA production (ng) / (2 x 324.5)

[0093] Note: 2 represents that double-stranded DNA is composed of two single-stranded DNAs; 324.5 is the relative average molecular weight of dNTP.

[0094] 3.6 Calculation of enzyme amount A (U) added in 25ul reaction system

[0095] Enzyme amount A (U) = dsDNA production (ng) x 3 / (2 x 324.5 x 10) = dsDNA production (ng) x 4.62 x 10-4.

[0096] Note: dsDNA production (ng) is the production in 10 minutes, x 3 represents the production in 30 minutes; / 10 represents that the amount of dNTP (nmol) consumed in 30 min is several times of 10 nmol;

[0097] 3.7 Calculation of mother liquor concentration

[0098] Mother liquor absolute activity (U / uL) = (enzyme amount A / 5) x total dilution factor, total dilution factor = mother liquor theoretical activity / enzyme loading gradient.

[0099] Note: 5 represents that 5uL of enzyme is added in the final reaction system; the total dilution factor is the dilution factor when the enzyme mother liquor is diluted to the final reaction system.

[0100] Four, analysis of antibody Taq DNA polymerase activity value results

[0101] According to the above method, the three repeated determination results of the activity of the tested enzyme 1 are 5.62 U / uL, 5.48 U / uL, and 5.76 U / uL, the average value is 5.62 U / uL, and the CV value is 2.43%; the three repeated determination results of the activity of the tested enzyme 2 are 5.93 U / uL, 5.44 U / uL, and 5.50 U / uL, the average value is 5.62 U / uL, and the CV value is 4.78%; the three repeated determination results of the activity of the tested enzyme 3 are 5.58 U / uL, 5.90 U / uL, and 5.54 U / uL, the average value is 5.67 U / uL, and the CV value is 3.48%; the three repeated determination results of the activity of the tested enzyme 4 are 5.94 U / uL, 5.93 U / uL, and 6.06 U / uL, the average value is 5.98 U / uL, and the CV value is 1.22%; the three repeated determination results of the activity of the tested enzyme 5 are 5.88 U / uL, 5.87 U / uL, and 6.33 U / uL, the average value is 6.03 U / uL, and the CV value is 4.32%.

[0102] The normality test of the five groups of Taq DNA polymerase activity value results obtained by real-time fluorescence quantitative PCR was performed by using IBM SPSS 26 software, and the results are shown in Table 7.

[0103] Table 7 Normality test of five groups of antibody Taq DNA polymerase activity value data

[0104]

[0105] Since the sample size is small, the K-S result is used as the reference, and the statistical calculation significance is greater than 0.05, which is subject to normal distribution. Further confirmation by viewing the Q-Q graph Figure 9 ) can be seen that the expected standard basically distributes up and down near the straight line, and it can be considered to be subject to normal distribution.

[0106] The Grubbs and Dixon outlier test methods were used to test the outliers of the five groups of antibody Taq DNA polymerase activity value data, and no outliers were found, and the results are shown in Table 8.

[0107] Table 8 Outlier test of five groups of antibody Taq DNA polymerase activity value data

[0108]

[0109] The final value of the five-tube enzyme activity was 5.62 U / uL, 5.62 U / uL, 5.67 U / uL, 5.98 U / uL, and 6.03 U / uL, respectively, as determined by the method. The CV value of each group of data was less than 5%, and combined with the normality test of the experimental results, the abnormal value detection and other analyses, it can be seen that the method has the characteristics of safety, convenience, rapidness, sensitivity, accuracy and good repeatability in the determination of antibody Taq DNA polymerase activity. The method has innovation and certain advancement compared with the existing method.

[0110] V. Effect of different concentrations of fluorescent dyes on the determination results

[0111] To explore the effect of fluorescent dye concentration on the determination results, different concentrations of EvaGreen fluorescent dye were designed for comparative experiments.

[0112] 5.1 Experimental design

[0113] Three final concentrations of 0.5x, 0.8x and 1.0x EvaGreen fluorescent dye were selected, and other conditions were the same as in Example 1. The activity of the same batch of antibody Taq DNA polymerase (No. 1) was determined. Each concentration condition was determined in triplicate.

[0114] 5.2 Experimental results and analysis

[0115] Under different concentrations of EvaGreen fluorescent dye, the activity of antibody Taq DNA polymerase was determined as follows:

[0116] 0.5x EvaGreen: 5.32 U / uL, 5.29 U / uL, 5.41 U / uL, average value 5.34 U / uL, CV value 1.15%

[0117] 0.8x EvaGreen: 5.62 U / uL, 5.48 U / uL, 5.76 U / uL, average value 5.62 U / uL, CV value 2.43%

[0118] 1.0x EvaGreen: 5.65 U / uL, 5.54 U / uL, 5.73 U / uL, average value 5.64 U / uL, CV value 1.69%

[0119] The results showed that the enzyme activity results determined by 0.8x and 1.0x EvaGreen fluorescent dye were similar, while the results determined by 0.5x EvaGreen were slightly lower. This may be because low concentration of fluorescent dye is not enough to completely bind to the newly generated double-stranded DNA, resulting in low fluorescence signal. Considering the cost of fluorescent dye and signal intensity, 0.8x EvaGreen was selected as the optimal concentration, which can ensure sufficient fluorescence signal and save reagent cost.

[0120] Six, the influence of different templates on the determination results

[0121] To verify the influence of template selection on the determination results, this embodiment compares the determination effects of M13 bacteriophage single-stranded DNA and short palindromic sequence used in the national standard method as templates.

[0122] 6.1 Experimental design

[0123] 200 ng / μL M13 mp18 single-stranded DNA and short palindromic sequence specified in the national standard method (GB / T 35542-2017) were used as templates respectively, and other conditions were the same as in Example 1. The activity of antibody Taq DNA polymerase (No. 1) in the same batch was determined. Each template condition was determined in triplicate.

[0124] 6.2 Experimental results and analysis

[0125] Under different template conditions, the activity of antibody Taq DNA polymerase was determined as follows:

[0126] M13 mp18 single-stranded DNA: 5.62 U / μL, 5.48 U / μL, 5.76 U / μL, average value 5.62 U / μL, CV value 2.43%

[0127] Short palindromic sequence: 5.24 U / μL, 4.98 U / μL, 5.71 U / μL, average value 5.31 U / μL, CV value 7.05%

[0128] The results show that the repeatability of the determination results using M13 mp18 single-stranded DNA as a template is significantly better than that using a short palindromic sequence. This verifies the advantages of using M13 filamentous bacteriophage single-stranded DNA as a template in the present application, overcoming the upper limit of fluorescence acquisition caused by the short palindromic sequence in the national standard method, and improving the accuracy and repeatability of the determination.

[0129] Seven, comparison between isotope labeling method and the method of the present application

[0130] To compare the differences between the method of the present application and the traditional isotope labeling method, the following comparative experiments were performed.

[0131] 7.1 Experimental design

[0132] The same batch of antibody Taq DNA polymerase (No. 1) was selected, and its activity was determined using the method of the present application and the isotope labeling method (³H-TTP incorporation method) respectively. Each method was determined in triplicate.

[0133] 7.2 Experimental results and analysis

[0134] The results of the antibody Taq DNA polymerase activity determined by the two methods are as follows:

[0135] The method of the present application: 5.62 U / μL, 5.48 U / μL, 5.76 U / μL, the average value is 5.62 U / μL, and the CV value is 2.43%;

[0136] Isotope labeling method: 5.85 U / μL, 5.73 U / μL, 5.91 U / μL, the average value is 5.83 U / μL, and the CV value is 1.56%;

[0137] The results determined by the two methods are similar, indicating that the accuracy of the method of the present application is comparable to that of the conventional isotope labeling method. However, the method of the present application has obvious safety and convenience advantages: no radioactive substances are used, avoiding the safety hazards and complex waste treatment of the isotope labeling method; the determination time is short, and the operation is simple; no special protective equipment is needed, reducing the experimental cost.

[0138] Eight, comparison between the national standard method and the method of the present application

[0139] In order to verify the advantages of the method of the present application relative to the national standard method (GB / T 35542-2017), the following comparative experiments were carried out.

[0140] 8.1 Experimental design

[0141] The same batch of antibody Taq DNA polymerase (Nos. 1 to 5) was selected, and the activity thereof was determined by the method of the present application and the national standard method, respectively. Each enzyme was determined by each method for 3 times.

[0142] 8.2 Experimental results and analysis:

[0143] The CV values of the five enzymes determined by the two methods are compared as follows:

[0144] Enzyme 1: the CV value of the method of the present application is 2.43%, and the CV value of the national standard method is 6.78%;

[0145] Enzyme 2: the CV value of the method of the present application is 4.78%, and the CV value of the national standard method is 8.22%;

[0146] Enzyme 3: the CV value of the method of the present application is 3.48%, and the CV value of the national standard method is 7.45%;

[0147] Enzyme 4: the CV value of the method of the present application is 1.22%, and the CV value of the national standard method is 5.89%;

[0148] Enzyme 5: the CV value of the method of the present application is 4.32%, and the CV value of the national standard method is 9.01%;

[0149] The results show that the repeatability of the method is obviously better than the national standard method, the CV values of all samples are less than 5%, and the CV values of the national standard method are all greater than 5%. This is mainly due to the fact that the template selection is optimized, the M13 filamentous bacteriophage single-stranded DNA is used instead of the short palindromic sequence in the national standard method, the limitation of the upper limit of fluorescence collection is overcome, the linear range and repeatability of the determination are improved. At the same time, the linear relationship between the dNTPs consumption and time is characterized, so that the determination method is more scientific and reasonable.

[0150] Through the detailed experimental verification of the above examples and comparative examples, the method of the present application has the following significant technical effects:

[0151] 1. Safe and convenient: Avoid the radioactivity hazard and complex waste treatment of the isotope labeling method, and the operation is safer and more convenient.

[0152] 2. Fast and sensitive: Through real-time fluorescent quantitative PCR technology, the DNA polymerization process is monitored in real time, the sensitivity is high, and the detection speed is fast.

[0153] 3. Accurate determination: The optimized template selection overcomes the limitation of the upper limit of fluorescence collection caused by the short palindromic sequence in the national standard method, the CV values of the determination results of the five enzymes are all less than 5%, which shows that the determination accuracy is high.

[0154] 4. Good repeatability: The optimized reaction conditions and calculation method establish a stable double standard curve system, statistical analysis shows that the determination results conform to the normal distribution and have no abnormal value, which proves that the method is reliable and stable.

[0155] 5. Low cost: No need to use expensive radioactive isotopes, reduce the determination cost.

[0156] 6. Suitable for high-throughput detection: The operation is simple, multiple samples can be processed at the same time, and it is suitable for high-throughput DNA polymerase activity detection requirements.

[0157] The present application successfully solves the safety, accuracy and repeatability problems in the prior art by optimizing the template selection, reaction conditions and calculation method, provides a safe, convenient and reliable new method for accurate determination of antibody Taq DNA polymerase activity, and has important practical application value.

Claims

1. A method for absolute determination of antibody Taq DNA polymerase activity based on real-time fluorescence quantitative detection, characterized in that, The method includes the following steps: a) Using double-stranded λDNA of different concentrations as standards, add saturated nucleic acid fluorescent dye, record fluorescence values, and construct a standard curve of double-stranded λDNA content versus fluorescence value; b) Using M13 phage single-stranded DNA as a template, the antibody Taq DNA polymerase was added to the reaction system containing M13R primers, dNTPs and saturated nucleic acid fluorescent dye to initiate the extension reaction, and the fluorescence values ​​at different time points were recorded in real time at 74℃. c) Calculate the net fluorescence value at different time points during the reaction process and establish a linear relationship between the net fluorescence value and the reaction time; d) Based on the standard curve established in step a), calculate the amount of newly generated double-stranded DNA at different time points in step c); e) Calculate the amount of dNTPs consumed based on the amount of newly generated double-stranded DNA; f) Calculate the absolute activity of the antibody Taq DNA polymerase according to the definition of antibody Taq DNA polymerase activity; The saturated nucleic acid fluorescent dye is EvaGreen fluorescent dye, with a final concentration of 0.8×. The M13R primer sequence in step b) is 5′-GTTGTAAAACGACGGCCAG-3′; The formula for calculating the dNTPs consumption in step e) is: dNTP consumption (nmol) = Amount of newly generated double-stranded DNA (ng) / (2 × 324.5), Where 2 represents that double-stranded DNA is composed of 2 single-stranded DNA molecules, and 324.5 is the relative average molecular weight of dNTPs; The formula for calculating the Taq DNA polymerase activity of the antibody to be tested in step f) is as follows: Enzyme quantity (U) = Amount of newly generated double-stranded DNA (ng) × 3 / (2 × 324.5 × 10) = Amount of newly generated double-stranded DNA (ng) × 4.62 × 10 -4 , The amount of newly generated double-stranded DNA is the amount generated within 10 minutes. Multiplying by 3 represents the amount generated within 30 minutes, and dividing by 10 represents the amount of dNTPs consumed within 30 minutes, which is several times that of 10 nmol.

2. The method according to claim 1, characterized in that, The concentration gradient of the double-stranded λDNA standard in step a) is 0, 5, 10, 25, 50, and 100 ng.

3. The method according to claim 1, characterized in that, The reaction system in step b) further includes: 1× activity assay buffer; 3mM MgCl2; 0.2mM dNTPs; 0.4μM 13R primers; 200ng / μL M13mp18 single-stranded DNA.

4. The method according to claim 1, characterized in that, The concentration of the antibody Taq DNA polymerase in step b) is 0.008 U / μL.

5. The method according to claim 1, characterized in that, The formula for calculating the absolute vitality of the mother liquor in step f) is: The absolute activity of the stock solution (U / μL) = (enzyme amount / 5) × total dilution factor. Where 5 represents the amount of enzyme added to the final reaction system, and the total dilution factor is the dilution factor when diluting from the enzyme stock solution to the final reaction system.

6. The method according to claim 1, characterized in that, The PCR reaction procedure in step b) includes: Three fluorescence readings were performed at 74℃ to serve as the initial values. Activate at 95℃ for 1 minute; 74℃, 30 seconds, 30 cycles, with one fluorescence signal collected per cycle.

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