A constant temperature nucleic acid amplification method and reaction system based on Dda helicase

Abstract

The present invention relates to the field of molecular biology technology, and specifically to a Dda helicase-based constant-temperature nucleic acid amplification method and reaction system, the reaction system comprising a core enzyme mixture, a buffer, an RNA modified primer mixture, a fluorescent detection probe mixture, an ATP / ADP mixture, and a dNTPs mixture; the core enzyme mixture comprises Dda helicase, MutS protein, RNase H2, and Bst DNA polymerase. A triple guarantee mechanism is established for MutS protein mismatch detection, RNA modified primer control, and RNase H2 specific cleavage, giving the nucleic acid amplification method excellent specificity, superb multiplex detection capabilities, and extremely high detection sensitivity. The nucleic acid amplification method is more convenient to operate and more suitable for standardized applications. The total reaction time is also shorter, and no complex temperature cycling is required, making it more suitable for on-site rapid detection. The method solves the problems of insufficient specificity and low amplification efficiency in existing constant-temperature nucleic acid amplification technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a constant-temperature nucleic acid amplification method and a reaction system based on Dda helicase. Background Art

[0002] Nucleic acid amplification (NAA) is a core technology in modern molecular biology. Among numerous nucleic acid amplification methods, polymerase chain reaction (PCR) has been widely used in biological research and clinical diagnosis due to its significant advantages, such as high sensitivity and specificity. However, PCR relies on precise temperature cycling equipment, which achieves DNA amplification through repeated heating and cooling cycles. This complex temperature control requirement not only increases equipment cost and operational difficulty, but also significantly limits its application in rapid on-site testing scenarios. Isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and transcription-mediated amplification (TMA) all enable rapid nucleic acid amplification under constant temperature conditions, eliminating the need for complex temperature cycling equipment. This greatly simplifies the operational process, reduces the requirements for the detection environment, and opens up new possibilities for rapid on-site testing. However, these isothermal amplification techniques still face several technical challenges that need to be addressed in practical applications. Specifically, due to their relatively low reaction temperatures, they are prone to nonspecific amplification, resulting in insufficient specificity and false-positive results. Furthermore, increased nonspecific amplification and primer dimers can lead to high limits of detection, compromising the detection of low-copy-number targets. These isothermal amplification techniques can only detect two to five targets simultaneously, making them difficult to meet the needs of multiplexed detection. Furthermore, competitive inhibition is prone to occur when both high- and low-abundance targets are present, resulting in a narrow dynamic range. Furthermore, primer design for these isothermal amplification techniques is complex; for example, LAMP requires six to eight primers. Primers are prone to dimerization, further impacting amplification efficiency.

[0003] Dda helicase (Deoxyribonucleic acid-dependent ATPase helicase) is a key enzyme in bacteriophage T4. It is a single-stranded DNA-dependent ATPase and belongs to the SF1 (Superfamily 1) helicase family. Dda helicase plays a central role in the DNA replication system of bacteriophages such as T4 and T6, particularly in unwinding double-stranded DNA and forming single-stranded DNA templates. It unwinds DNA in the 5'→3' direction, exhibiting extremely high unwinding efficiency and unique unwinding capabilities, particularly for DNA with high GC content and complex secondary structure. This unique unwinding property makes Dda helicase extremely promising for application in the cutting-edge field of isothermal amplification. However, the practical application of isothermal amplification technology still faces many challenges in using Dda helicase solely as an unwinding tool, particularly the issue of specificity control. The MutS protein is a key component of the DNA mismatch repair system, specifically recognizing and binding to DNA mismatches to block DNA polymerase elongation, thereby effectively preventing erroneous DNA replication and amplification. RNase H2 is a specific ribonuclease that specifically cleaves the RNA strand within DNA / RNA hybrids, playing a crucial role in maintaining the stability and specificity of nucleic acid structures. Although Dda helicase, MutS protein, and RNase H2 each possess unique functions and value in nucleic acid amplification processes, there are currently no reports on combining these three substances for isothermal nucleic acid amplification technology.

[0004] The present invention provides a constant-temperature nucleic acid amplification method and reaction system based on Dda helicase, so as to solve the problems of insufficient specificity and low amplification efficiency of the existing constant-temperature nucleic acid amplification technology in the prior art. Summary of the Invention

[0005] The purpose of the present invention is to provide a constant temperature nucleic acid amplification method and reaction system based on Dda helicase to solve the problems of insufficient specificity and low amplification efficiency of the existing constant temperature nucleic acid amplification technology in the prior art.

[0006] The technical solution of the present invention is: a reaction system for isothermal nucleic acid amplification based on Dda helicase, the reaction system comprising a core enzyme mixture, a buffer, an RNA modified primer mixture, a fluorescent detection probe mixture, an ATP / ADP mixture, and a dNTPs mixture;

[0007] The core enzyme mixture includes Dda helicase, MutS protein, RNase H2 and Bst DNA polymerase; the Dda helicase, the MutS protein and the RNase H2 cooperate with each other to form a triple protection mechanism.

[0008] Preferably, in the reaction system, the concentration of the Dda helicase is 2-5 U / μL, the concentration of the MutS protein is 0.5-1.2 μM, the concentration of the RNase H2 is 0.2-0.5 U / μL, and the concentration of the Bst DNA polymerase is 8-16 U / μL.

[0009] Preferably, the Dda helicase is a DNA helicase derived from thermophilic bacteria and has the activity of unwinding double-stranded DNA in the temperature range of 37 to 50°C.

[0010] Preferably, the MutS protein is any one of the Escherichia coli MutS protein, a functional mutant of the Escherichia coli MutS protein, a human MutSα protein, and a functional mutant of the human MutSα protein.

[0011] Preferably, in the RNA modified primer mixture, the structure of the RNA modified primer is 5'-DNA sequence 1-ribonucleotide-DNA sequence 2-3'; wherein the ribonucleotide is any one or more of rA, rU, rG, and rC;

[0012] The DNA sequence 1 is 8 to 15 nucleotides in length; the DNA sequence 2 is 8 to 15 nucleotides in length;

[0013] There are 8 to 12 nucleotides between the ribonucleotide and the 3' end.

[0014] Preferably, the buffer comprises Tris-HCl, MgCl2, and KCl; the pH of the Tris-HCl is 7.8 to 8.2;

[0015] In the reaction system, the concentration of Tris-HCl is 20-30 mM, the concentration of MgCl2 is 6-12 mM, and the concentration of KCl is 50-100 mM.

[0016] Preferably, in the reaction system, the concentration of ATP is 1.0-2.0 mM, and the concentration of ADP is 0.1-0.3 mM.

[0017] Preferably, the reaction system further comprises a stabilizing component; the stabilizing component is any one or more of betaine, BSA, trehalose, and DTT.

[0018] The present invention also provides a constant temperature nucleic acid amplification method based on Dda helicase, comprising the following steps:

[0019] S1. Pre-treating the sample to release and dilute the nucleic acid molecules in the sample to form a sample to be tested;

[0020] S2. Add the sample to be tested to the above reaction system, perform a staged isothermal amplification reaction at 30-42°C, and monitor the changes in the fluorescence signal in real time during the amplification process. After 15-50 minutes, the nucleic acid amplification is completed.

[0021] Preferably, the staged constant temperature amplification reaction includes three stages: the first stage is a warm amplification reaction at 30-37°C, with a reaction time of 2-5 minutes; the second stage is a warm amplification reaction at 37°C, with a reaction time of 5-10 minutes; the third stage is a warm amplification reaction at 37-42°C, with a reaction time of 15-30 minutes.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] (1) The present invention provides a constant temperature nucleic acid amplification method and reaction system based on Dda helicase. The core enzyme mixture in the reaction system includes Dda helicase, MutS protein, RNase H2 and Bst DNA polymerase. Through the synergistic effect between Dda helicase, MutS protein and RNase H2, MutS protein mismatch detection, RNA modified primer control and RNase H2 are constructed. The triple guarantee mechanism of H2-specific cleavage gives the nucleic acid amplification method excellent specificity, and its specificity reaches more than 99.9%, which can effectively eliminate the occurrence of false positive results; the nucleic acid amplification method can simultaneously detect more than 50 targets in a single reaction tube, and has super strong multiple detection capabilities; at the same time, the nucleic acid amplification method also has extremely high detection sensitivity, and its detection limit is as low as 1 to 10 copies, reaching the level of single-molecule detection, greatly improving the detection capability of trace samples; in addition, the operation of the nucleic acid amplification method is more convenient, and its reagents are all pre-mixed in form, and no complicated preparation process is required when in use, which is more suitable for automated and standardized applications; the total reaction time of the nucleic acid amplification method is shorter, and no complicated temperature cycle is required, which is suitable for on-site rapid detection; it solves the problems of insufficient specificity and low amplification efficiency of the existing constant temperature nucleic acid amplification technology in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0025] Figure 1 The SDS-PAGE protein electrophoresis detection result of the target protein of the present invention after affinity chromatography;

[0026] Figure 2 Schematic diagram of the triple specificity control mechanism of the reaction system of the present invention when detecting sample DNA;

[0027] Figure 3This is the amplification curve of the reaction system described in the first embodiment of the present invention when there is no sample DNA;

[0028] Figure 4 This is the amplification curve of the reaction system in the second embodiment of the present invention when there is no sample DNA;

[0029] Figure 5 This is the amplification curve of the reaction system described in the first embodiment of the present invention when detecting sample DNA;

[0030] Figure 6 This is the amplification curve of the reaction system described in the third embodiment of the present invention when detecting sample DNA;

[0031] Figure 7 This is the amplification curve of the reaction system described in the fourth embodiment of the present invention when detecting sample DNA;

[0032] Figure 8 The amplification curves of the reaction systems described in embodiments five to seven of the present invention when detecting sample DNA;

[0033] Figure 9 This is the amplification curve of the reaction system described in the third embodiment of the present invention when detecting 2 to 20 copies of the test sample DNA;

[0034] Figure 10 The figure shows the amplification curve when a commercial qRT-PCR kit is used to detect 2 to 20 copies of the test sample DNA. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to specific embodiments:

[0036] Dda helicase is a monomeric protein with a molecular weight of approximately 55 kDa. It is not a multi-subunit complex, but rather achieves its unwinding function through a single protein chain. Dda helicase has an ATPase domain that provides energy through ATP hydrolysis to drive DNA unwinding. Dda helicase also contains a dewinding active domain that interacts with DNA and unwinds its double-stranded structure. In the following embodiments, Dda helicase is selected from a DNA helicase derived from thermophilic bacteria.

[0037] 1. Cloning and Expression of Dda Helicase

[0038] A specialized company (General Bio (Anhui) Co., Ltd.) was commissioned to extract the amino acid sequence of a DNA helicase from a thermophilic bacterium and synthesize the required DNA helicase gene sequence in large quantities through chemical synthesis. The amino acid sequence of the DNA helicase is shown in SEQ ID No. 1. Subsequently, the DNA helicase gene sequence was precisely inserted into the pET-28 expression vector to construct a vector carrying the DNA helicase gene sequence (designated pET28a), ultimately producing pET28a plasmid vector powder. The resulting pET28a plasmid vector powder was then transformed into SHuffle T7 cells. SHuffle T7 cells, an optimized E. coli expression system, express the recombinant protein encoded by the DNA helicase gene sequence at high levels under the control of the T7 promoter in the pET-28 vector. Furthermore, the unique properties of SHuffle T7 cells promote the correct folding of disulfide-bonded proteins, thereby ensuring their biological activity and stability.

[0039] First, prepare the culture medium by weighing 12g of tryptone, 24g of yeast extract, 2.31g of KH2PO4, 12.54g of K2HPO4, and 4mL of glycerol (0.4% v / v) into a beaker, dissolving them in deionized water and adjusting the volume to 1L to form a mixture. Adjust the pH of the mixture to 7.0-7.2 using 1M NaOH or 1M HCl. Add additives such as kanamycin, MgSO4·7H2O, and a trace element solution to a final concentration of 50μg / mL kanamycin, 1mM MgSO4, and 1mL / L of the trace element solution to obtain the culture medium. Next, inoculate the transformed SHuffle T7 cells into the culture medium and culture with shaking at 37°C and 220 rpm for 6-8 hours to allow the cells to grow to the logarithmic phase. Afterwards, add IPTG to the culture system to a final concentration of 1mM, set the induction temperature to 18-22°C, and continue shaking culture for 8-12 hours to induce target protein expression. After induction, transfer the culture medium to a centrifuge tube and centrifuge at 4°C, 6000×g for 10 minutes to collect the bacterial pellet; then wash the bacterial pellet twice with pre-chilled PBS buffer. After each wash, centrifuge the bacterial pellet at 4°C, 6000×g for 10 minutes and discard the supernatant to remove residual culture medium components.

[0040] Prepare a disruption buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl, 10mM imidazole, 5% glycerol, and 0.1% Triton X-100. Before use, add PMSF and protease inhibitors to the disruption buffer, adjusting the concentration of PMSF to 1mM and the protease inhibitor cocktail to 1x to prevent protein degradation. Resuspend the washed bacterial pellet in an appropriate amount of disruption buffer and disrupt the pellet using a high-pressure homogenizer at a pressure of 800-1200 bar and 2-3 passes. Precool the equipment and materials to 4°C during the disruption process, and control the flow rate to ensure the temperature rise does not exceed 10°C. The disrupted bacterial suspension was centrifuged at 4°C and 2000×g for 30 minutes to precipitate the cell debris. The supernatant was collected and filtered through a 0.45 μm filter to remove residual cell debris and impurities to obtain a clarified supernatant for subsequent target protein purification steps.

[0041] 2. Purification of target protein

[0042] Prepare a binding buffer (Buffer A) containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM imidazole, 5% glycerol, and 0.1% Tween-20 and set aside. Tween-20 is included in the binding buffer to reduce nonspecific binding of proteins in the supernatant to nickel ions on the Ni-NTA resin. Prepare a wash buffer (Buffer B) containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, and 5% glycerol and set aside. Prepare an elution buffer (Buffer C) containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10-250 mM imidazole, 5% glycerol, and 1 mM DTT and set aside. DTT is included in the elution buffer to create a reducing environment.

[0043] Calculate the required resin volume based on the estimated amount of total protein in the sample to be processed. Typically, 1 mL of resin can process approximately 50-100 mg of total protein. Select an affinity chromatography column (Ni-NTA column) with an appropriate column volume.

[0044] First, rinse the affinity chromatography column with 5 column volumes of binding buffer to stabilize the column bed volume; during the rinsing process, closely observe the baseline of the UV detector to ensure that the baseline is stable, so that the resin in the chromatography column is fully balanced and reaches a stable state, ready for subsequent sample loading. Then slowly load the filtered supernatant onto the chromatography column, and control the loading flow rate to 0.5-1.0 mL / min to allow the target protein in the supernatant to specifically bind to the nickel ions on the Ni-NTA resin. After the loading is completed, first rinse the affinity chromatography column with 5 column volumes of binding buffer to remove unbound impurity proteins; then wash the affinity chromatography column with 5 column volumes of wash buffer to further remove non-specifically bound proteins; and continuously monitor the absorbance changes of the target protein at a wavelength of 280 nm until it stabilizes at the baseline level. Finally, 20 column volumes of elution buffer containing 10-250 mM imidazole were used for gradient elution, and the elution peaks were collected. The results of SDS-PAGE protein electrophoresis showed that Figure 1 As shown, the elution buffer containing 40 mM imidazole elutes a significant amount of the target protein, Dda helicase. Alternatively, a gradient elution can be performed using elution buffers containing 5 column volumes of 50 mM, 100 mM, and 250 mM imidazole, respectively, and the elution peaks collected. Transfer the purified target protein solution to a clean centrifuge tube, add glycerol to a final concentration of 40%, and store the aliquots at -80°C to avoid repeated freeze-thaw cycles.

[0045] In order to make the chromatography column reusable, the resin needs to be regenerated. First, flush the column with 5 column volumes of 0.5M NaOH solution at a flow rate of 0.5-1.0 mL / min to remove residual proteins, impurities, and possible microorganisms on the resin surface. Then, flush the column with deionized water at the same flow rate until the pH value of the effluent is close to neutral to remove residual NaOH solution. After that, equilibrate the column with Buffer A to restore the resin to its initial state and store it at 4°C for next use.

[0046] Implementation Method 1

[0047] 1. Prepare a reaction system using the target protein obtained above;

[0048] 1. Preparation of buffer solution;

[0049] 1.21 g of Tris-HCl (pH 8.0), 0.203 g of MgCl₂·6H₂O, and 0.373 g of KCl were weighed and added to a beaker. The volume was adjusted to 10 mL with sterile water to obtain a buffer solution with a final concentration of 200 mM Tris-HCl, 100 mM MgCl₂, and 500 mM KCl. In other embodiments, a buffer solution with a final concentration of 200-300 mM Tris-HCl, 60-120 mM MgCl₂, and 500-1000 mM KCl can be prepared, such that the concentrations of Tris-HCl, MgCl₂, and KCl in the entire reaction system are 20-30 mM, 6-12 mM, and 50-100 mM, respectively.

[0050] 2. Preparation of ATP / ADP mixed solution

[0051] Weigh 46.1 mg of ATP·Na3 and 5.5 mg of ADP·Na2 into a beaker, dissolve them in 10 mM Tris-HCl (pH 7.5), and dilute to 5 mL. This yields an ATP / ADP mixture with a final concentration of 15 mM ATP·Na3 and 2 mM ADP·Na2. In other embodiments, the ATP / ADP mixture can be prepared with a final concentration of 10-20 mM ATP·Na3 and 1-3 mM ADP·Na2, resulting in an ATP concentration of 1.0-2.0 mM and an ADP concentration of 0.1-0.3 mM in the reaction system. By regulating the ATP and ADP concentrations in the reaction system, the activity of the Dda helicase can be precisely controlled.

[0052] 3. Preparation of dNTPs mixture

[0053] Mix equal volumes of dATP, dTTP, dGTP, and dCTP, all at a concentration of 10 mM, to obtain a dNTPs mixture. In other embodiments, the concentrations of dATP, dTTP, dGTP, and dCTP in the reaction system should all be within the range of 0.8 to 1.6 mM.

[0054] 4. Preparation of core enzyme mixture

[0055] Take appropriate amounts of DDa helicase, RNase H2, and Bst polymerase, and use enzyme diluent containing 20% ​​glycerol to prepare a core enzyme mixture with a DDa helicase concentration of 160 U / μL, a MutS protein concentration of 32 μM, an RNase H2 concentration of 12 U / μL, and a Bst polymerase concentration of 480 U / μL. In other embodiments, an enzyme diluent containing 20% ​​glycerol can also be used to prepare a core enzyme mixture with a DDa helicase concentration of 40-320 U / μL, a MutS protein concentration of 0-48 μM, an RNase H2 concentration of 8-20 U / μL, and a Bst polymerase concentration of 320-640 U / μL; thereby, in the entire reaction system, the concentration of DDa helicase is 1-8 U / μL, the concentration of MutS protein is 0-1.2 μM, the concentration of RNase H2 is 0.2-0.5 U / μL, and the concentration of Bst polymerase is 8-16 U / μL.

[0056] 5. Preparation of stabilizing components

[0057] Betaine is prepared at a concentration of 5 M as a stabilizing component. In other embodiments, the stabilizing component can also be any one or more of betaine, BSA, trehalose, DTT, etc. In addition, in the reaction system, the concentration of betaine needs to be controlled within the range of 0.5 to 1.5 M; the concentration of BSA needs to be controlled within the range of 0.05 to 0.3 mg / mL; the concentration of trehalose needs to be controlled within the range of 50 to 150 mM; and the concentration of DTT needs to be controlled within the range of 0.5 to 2.0 mM. Alternatively, no stabilizing component can be added to the reaction system.

[0058] 6. Primer design

[0059] RNA modified primers were designed using the SARS-CoV-2 N gene as sample DNA (target); the partial target sequence, primer, and probe designs are shown in Table 1; and the RNA modified primers and probes in Table 1 were respectively prepared into an RNA modified primer mixture and a fluorescent probe mixture for use, wherein the concentration of each RNA modified primer in the RNA modified primer mixture was 20 μM; and the concentration of each fluorescent detection probe was 6 μM.

[0060] In other embodiments, the structure of the RNA modified primer is 5'-DNA sequence 1-ribonucleotide-DNA sequence 2-3'; wherein the ribonucleotide is any one or more of rA, rU, rG, rC, etc.; the length of DNA sequence 1 is 8 to 15 nucleotides; the length of DNA sequence 2 is 8 to 15 nucleotides; there is an interval of 8 to 12 nucleotides between the ribonucleotide and the 3' end; in the RNA modified primer mixture, the concentration of each RNA modified primer needs to be controlled in the range of 2 to 20 μM, and thus the concentration of each RNA modified primer in the entire reaction system is within the range of 0.2-2.0 μM. In the reaction system, the concentration of each fluorescent detection probe needs to be controlled in the range of 0.1 to 0.5 μM.

[0061] Table 1. Sequences of targets, RNA modification primers, and probes

[0062]

[0063] A reaction system was prepared by combining 2.0 μL of the prepared buffer, 2.0 μL of the ATP / ADP mixture, 2.8 μL of the dNTPs mixture, 3.2 μL of betaine, 1.0 μL of the RNA-modified primer mixture, 0.5 μL of the core enzyme mixture, 6.5 μL of sterile water, and 1.0 μL of the fluorescent probe mixture. 1.0 μL of the sample DNA to be tested was then added to the reaction system for detection. Simultaneously, in the absence of a sample DNA template, the amplification status of the reaction system was monitored in real time.

[0064] The specific detection method includes: S1, pre-treating the sample DNA to release the nucleic acid molecules in the sample DNA and diluting it to an appropriate concentration to form the sample DNA to be detected;

[0065] S2. Add 1.0 μL of the sample DNA to be tested to the above reaction system and perform a staged isothermal amplification reaction at 30-42°C. Monitor the changes in the fluorescence signal in real time during the amplification process. After 25 minutes, the nucleic acid amplification is completed. The staged isothermal amplification reaction consists of three stages: the first stage is a warm amplification reaction at 32°C for 4 minutes; the second stage is a warm amplification reaction at 37°C for 8 minutes; and the third stage is a warm amplification reaction at 40°C for 23 minutes. Monitor the changes in the fluorescence signal in real time during the amplification process, and determine the presence and concentration of the target based on the signal intensity and appearance time.

[0066] like Figure 2As shown, the purpose of the first and second stage isothermal amplification reactions is to encourage Dda helicase to open the double-stranded DNA structure and pair the RNA modified primer with the single-stranded DNA; at the same time, it encourages the MutS protein to scan and bind to the mismatch site on the sample DNA, thereby inhibiting the extension reaction of the DNA polymerase and blocking nonspecific amplification. Figure 2 The purpose of the third stage of warm amplification reaction is to allow the RNase H2 enzyme to recognize the RNA site in the double-stranded DNA, accurately cut and remove the RNA fragment, and form a gap structure at the cut site; Bst DNA polymerase recognizes this gap site and performs an extension reaction, while performing strand displacement to release a single-stranded DNA; at this point, two new double-stranded DNAs and two single-stranded DNAs will be generated in the reaction system, as shown in the following figure. Figure 2 Steps 3, 4, and 5 in the original text are repeated in this stage. At this stage, the newly generated double-stranded DNA will repeat the process of steps 1 to 2. At the same time, the upstream and downstream RNA modified primers will bind to the released single-stranded DNA respectively, and synthesize new double-stranded DNA molecules under the action of polymerase. The RNase H2 enzyme will recognize the RNA site on the newly formed double-stranded DNA again, and perform a cleavage reaction to form a new gap. The Bst DNA polymerase will recognize the gap and perform an extension reaction to form a complete double-stranded DNA while replacing the new single-stranded DNA. Figure 2 Then, Dda helicase unwinds the double-stranded structure, and the entire reaction cycle is repeated, achieving exponential amplification under constant temperature conditions.

[0067] Implementation Method 2

[0068] The difference between this embodiment and the first embodiment is that: a reaction system without MutS protein in the core enzyme mixture is selected; and the amplification status in the reaction system is monitored in real time in the absence of a sample DNA template.

[0069] Implementation Method 3

[0070] The difference between this embodiment and the first embodiment is that a reaction system with a MutS protein concentration of 0.5 μM in the core enzyme mixture is selected to detect the sample DNA.

[0071] Implementation Method 4

[0072] The difference between this embodiment and the first embodiment is that a reaction system with a MutS protein concentration of 1.5 μM in the core enzyme mixture is selected to detect the sample DNA.

[0073] Implementation Method Five

[0074] The difference between this embodiment and the first embodiment is that a reaction system with a DDa helicase concentration of 1 U / μL in the core enzyme mixture is selected to detect the sample DNA.

[0075] Implementation Method 6

[0076] The difference between this embodiment and the first embodiment is that a reaction system with a DDa helicase concentration of 2 U / μL in the core enzyme mixture is selected to detect the sample DNA.

[0077] Implementation Method Seven

[0078] The difference between this embodiment and the first embodiment is that a reaction system with a DDa helicase concentration of 8 U / μL in the core enzyme mixture is selected to detect the sample DNA.

[0079] Comparative Example 1

[0080] The sample DNA was detected using a commercial qRT-PCR kit.

[0081] During the detection process, the changes in the fluorescence signals in the reaction systems of Embodiments 1 to 7 are respectively monitored in real time, and the presence and concentration of the target are determined based on the signal intensity and appearance time.

[0082] Comparing the first embodiment with the second embodiment, it can be seen that Figure 3 、 Figure 4 As shown, a large amount of non-specific amplification was generated in the reaction system without the addition of MutS protein in the absence of DNA template; this further indicates that the introduction of MutS protein into the reaction system can block its non-specific amplification by binding to the mismatch sites of the sample DNA, making the nucleic acid amplification method have excellent specificity, and the specificity can reach more than 99.9%.

[0083] Comparing the first embodiment, the third embodiment, the fourth embodiment and the comparative example 1, it can be seen that Figure 5 、 Figure 6 、 Figure 7 As shown in the figure, when the concentration of MutS protein in the core enzyme mixture is 0.5uM, the amplification efficiency is optimal. However, when the concentration of MutS protein in the core enzyme mixture increases, the amplification efficiency does not increase significantly, but decreases instead. This further indicates that the concentration of MutS protein in the core enzyme mixture needs to be controlled at around 0.5uM.

[0084] Comparing the first embodiment, the fifth to seventh embodiments and the comparative example 1, it can be seen that Figure 8As shown, when the concentration of Dda helicase in the core enzyme mixture is lower than 4 U / uL, the amplification efficiency is low, while when the concentration of Dda helicase in the core enzyme mixture is in the range of 4 U / uL to 8 U / uL, the reaction efficiency is high.

[0085] 325 clinical respiratory samples were collected, including pharyngeal swabs, sputum, and alveolar lavage fluid, etc.; the pharyngeal swab samples were eluted with a nucleic acid releaser and used directly after elution. The reaction system prepared in Implementation Method Three was used to detect these 325 clinical respiratory samples; at the same time, a commercial qRT-PCR kit was used to detect these 325 clinical respiratory samples. The test results of the reaction system prepared in Implementation Method Three were as follows: the positive coincidence rate was 98.5% (196 / 199), the negative coincidence rate was 99.2% (125 / 126), the total coincidence rate was 98.8% (321 / 325), and the Kappa value was 0.97. At the same time, the average reaction time for detecting these 325 clinical respiratory samples using the reaction system prepared in Implementation Method Three was 20 minutes / sample; the average reaction time for detecting these 325 clinical respiratory samples using the commercial qRT-PCR kit was 120 minutes / sample. It can be seen from this that when the reaction system provided in this application is used to detect clinical respiratory samples, it not only has high accuracy, specificity, and excellent sensitivity, but also has the advantages of short detection time and suitability for on-site rapid detection.

[0086] The sample DNA was diluted into 2, 5, 10, and 20 copies of the DNA sample to be tested. The reaction system provided in the third embodiment and the commercial qRT-PCR kit were used to detect 2, 5, 10, and 20 copies of the DNA sample to be tested, respectively. Figure 9 、 Figure 10 As shown, the reaction system provided in the third embodiment has a high amplification effect on these 2, 5, 10, and 20 copies of the sample DNA to be tested; further illustrating that the reaction system provided by the present invention can construct a triple protection mechanism of MutS protein mismatch detection, RNA modified primer control, and RNase H2 specific cleavage through the synergistic effect between Dda helicase, MutS protein, and RNase H2, so as to reliably detect extremely low concentrations of target nucleic acid in the sample, so that the sensitivity of the detection method is sufficient to identify 1 to 10 target molecules; while the sensitivity of commercial qRT-PCR kits is not enough to identify 1 to 10 target molecules.

[0087] Specific RNA modified primers and probes are designed for each DNA or cDNA template to be tested and formulated to form a corresponding RNA modified primer mixture and fluorescent detection probe mixture. This RNA modified primer mixture and fluorescent detection probe mixture are combined with the same core enzyme mixture, buffer, ATP / ADP mixture, dNTPs mixture, and stabilization component as in Embodiment 3 to form a reaction system for simultaneously detecting 50 or more targets. The test results show that the reaction system provided by the present invention can simultaneously detect more than 50 targets in a single reaction through the synergistic action of Dda helicase, MutS protein, and RNase H2, achieving highly specific multiplex detection; and the total reaction time 3 does not exceed 40 minutes.

[0088] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they are not intended to limit the scope of protection of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

Claims

1. A constant temperature nucleic acid amplification reaction system based on Dda helicase, characterized in that: The reaction system includes core enzyme mixture, buffer, RNA modification primer mixture, fluorescent detection probe mixture, ATP / ADP mixture, and dNTPs mixture; The core enzyme mixture includes Dda helicase, MutS protein, RNase H2 and Bst DNA polymerase; the Dda helicase, the MutS protein and the RNase H2 cooperate with each other to form a triple protection mechanism; in the reaction system, the concentration of the Dda helicase is 4-8 U / μL, the concentration of the MutS protein is 0.5-1.2 μM, the concentration of the RNase H2 is 0.2-0.5 U / μL, and the concentration of the Bst DNA polymerase is 8-16 U / μL; In the RNA modified primer mixture, the structure of the RNA modified primer is 5'-DNA sequence 1-ribonucleotide-DNA sequence 2-3'; wherein the ribonucleotide is any one or more of rA, rU, rG, and rC; The DNA sequence 1 is 8 to 15 nucleotides in length; There are 8 to 12 nucleotides between the ribonucleotide and the 3' end.

2. The constant temperature nucleic acid amplification reaction system based on Dda helicase according to claim 1, characterized in that: The Dda helicase is a DNA helicase derived from thermophilic bacteria and has the activity of unwinding double-stranded DNA within the temperature range of 37 to 50°C.

3. The constant temperature nucleic acid amplification reaction system based on Dda helicase according to claim 1, characterized in that: The MutS protein is Escherichia coli MutS protein or human MutSα protein.

4. The constant temperature nucleic acid amplification reaction system based on Dda helicase according to claim 1, characterized in that: The buffer solution includes Tris-HCl, MgCl2, and KCl; the pH of the Tris-HCl is 7.8 to 8.2; In the reaction system, the concentration of Tris-HCl is 20-30 mM, the concentration of MgCl2 is 6-12 mM, and the concentration of KCl is 50-100 mM.

5. The constant temperature nucleic acid amplification reaction system based on Dda helicase according to claim 1, characterized in that: The ATP / ADP mixed solution includes ATP·Na3 and ADP·Na2; In the reaction system, the concentration of the ATP·Na3 is 1.0-2.0 mM, and the concentration of the ADP·Na2 is 0.1-0.3 mM.

6. The constant temperature nucleic acid amplification reaction system based on Dda helicase according to claim 1, characterized in that: The reaction system further includes a stabilizing component; the stabilizing component is any one or more of betaine, BSA, trehalose, and DTT.

7. A non-diagnostic constant temperature nucleic acid amplification method based on Dda helicase, characterized in that: The following steps are involved: S1. Pre-treating the sample to release and dilute the nucleic acid molecules in the sample to form a sample to be tested; S2. Add the sample to be tested to the reaction system of any one of claims 1 to 6, perform a staged isothermal amplification reaction at 30 to 42° C., and monitor the change in the fluorescence signal in real time during the amplification process. After 15 to 50 minutes, the nucleic acid amplification is completed. The staged constant temperature amplification reaction includes three stages: the first stage is a warm amplification reaction at 30-37°C, with a reaction time of 2-5 minutes; the second stage is a warm amplification reaction at 37°C, with a reaction time of 5-10 minutes; and the third stage is a warm amplification reaction at 37-42°C, with a reaction time of 15-30 minutes.

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