Enzyme combination for nucleic acid isothermal amplification reaction and use thereof

Abstract

The application provides an enzyme combination product for nucleic acid isothermal amplification reaction and application thereof, the recombinant enzyme combination product comprises a recombinant protein RecAm and a helicase UvrDm; the recombinant protein RecAm has an amino acid sequence as shown in SEQ ID NO. 2; the helicase UvrDm has an amino acid sequence as shown in SEQ ID NO. 4. The application screens RecA recombinant protein and helicase UvrD by molecular biology and bioinformatics methods, and the screened RecAm recombinant protein and helicase UvrDm can improve the affinity to DNA, reduce the sensitivity to pH and the dependence on Mg 2+ concentration when used in nucleic acid isothermal amplification reaction.

Description

Technical Field

[0001] This application relates to the field of molecular biotechnology, and in particular to an enzyme combination product for isothermal amplification of nucleic acids and its application. Background Technology

[0002] Nucleic acid amplification technology is an extremely important technique in modern life sciences. Polymerase chain reaction (PCR) is a typical example of nucleic acid amplification technology. Invented in 1985 by MμLlis et al. at the Human Genetics Laboratory of PE-Cetus in the United States, this technique uses linear DNA as a template for replication. However, this classic technique requires a temperature cycling instrument to unwind the double-stranded DNA, followed by cooling and annealing, primer pairing with the template strand, and finally primer extension at 72°C. This process is repeated to exponentially amplify the DNA. The entire PCR reaction process requires precise temperature control, which is expensive, requires highly skilled operators, and is only available in laboratories or medical institutions in some developed regions, significantly limiting the application of PCR-based molecular diagnostic techniques in rapid on-site diagnosis.

[0003] Isothermal amplification of nucleic acids (AFA) technology can achieve exponential amplification of target nucleic acid fragments without temperature-changing steps, greatly simplifying instrument requirements, eliminating the need for rigorously trained professionals, and reducing limitations on application scenarios. It also significantly accelerates the reaction time of nucleic acid amplification. Therefore, isothermal amplification technology better meets the "fast and convenient" requirements of modern molecular detection techniques and has significant value for field applications. In recent years, with the rapid development of molecular biology techniques, diagnostic methods based on isothermal amplification technology have been widely established and applied in laboratory testing for human diseases. Examples include chain substitution amplification (SDA) technology widely used by Becton Dickinson and Company in the diagnosis of tuberculosis, respiratory diseases, and sexually transmitted diseases; rolling circle amplification (RCA) technology used by Qiagen NV; helicase-dependent amplification (HDA) technology used by Quidel Corporation; single-primer isothermal amplification (SPIA) technology used by Tecan Genomics; and NEAR technology used in Abbott's ID NOW test kit, all of which have attracted considerable attention. In addition, there are loop-mediated isothermal amplification (LAMP) technology used by companies such as Bio-Rad Laboratories and Bio-Rad Laboratories for the detection of respiratory pathogens; recombinase-mediated strand replacement amplification (RAA) technology used by Zhejiang Taijing Biotechnology Co., Ltd. and Jiangsu Qitian Gene Co., Ltd.; SAT technology used by Shanghai Rendu Biotechnology Co., Ltd.; CPA technology used by Hangzhou Youstar Co., Ltd.; and recombinase polymerase amplification (RPA) technology used by TwistDx.

[0004] Among them, recombinase-mediated strand substitution amplification (RAA) technology amplifies the target fragment under room temperature conditions by simulating in vivo DNA amplification. This technology mainly relies on E. coli RecA recombinant protein, single-stranded DNA binding protein SSB, and strand substitution DNA polymerase. It has the advantages of high sensitivity, strong specificity, simple operation, short reaction time, no need for precise temperature control equipment, and no restrictions on professional personnel and application sites. However, this technology itself also has some defects and shortcomings: (1) The activity of recombinant protein RecA is inhibited by its own C-terminal amino acid residues, resulting in its weak affinity for ssDNA; (2) Recombinant protein RecA has a slow rate of ATP hydrolysis, and the ATP hydrolysis enzyme is incomplete; (3) In addition to using E. coli recombinant protein RecA, yeast protein Rad51 is also used in RAA technology, which increases the reaction cost; (4) The RAA reaction system contains recombinant protein RecA, yeast protein Rad51, single-stranded DNA binding protein, creatine phosphokinase, Bsu DNA polymerase and other proteases have high requirements for enzyme preservation solutions. A glycerol concentration of less than 50% will lead to a decrease in enzyme activity, which is very unfavorable for enzyme preservation and application. High glycerol enzymes require a high investment in freeze-drying applications. (5) RecA protein is sensitive to pH. Changes in pH can easily affect its activity, and a higher concentration of Mg is required. 2+ Only then can the activity of RecA be activated. All of the above factors limit the application of RAA. Summary of the Invention

[0005] Based on this, this application provides an enzyme combination product for nucleic acid isothermal amplification technology and its application.

[0006] According to one aspect of this application, an enzyme combination product for isothermal amplification of nucleic acids is provided, comprising a recombinant protein RecAm and a helicase UvrDm; said recombinant protein RecAm having an amino acid sequence as shown in SEQ ID NO.2; said helicase UvrDm having an amino acid sequence as shown in SEQ ID NO.4.

[0007] In one embodiment, the nucleotide sequence encoding the recombinant protein RecAm is shown in SEQ ID NO.1.

[0008] In one embodiment, the nucleotide sequence encoding the helicase UvrDm is shown in SEQ ID NO.3.

[0009] In one embodiment, it also includes one or more of a single-stranded DNA-binding protein, Bsu DNA polymerase, and creatine phosphokinase.

[0010] A kit for isothermal amplification of nucleic acids, comprising the enzyme combination product described above.

[0011] In one embodiment, it also includes one or more of Tris-HCl buffer, potassium chloride, polyethylene glycol 35000, dithiothreitol, dNTPs, ATP, creatine phosphate, forward primer, reverse primer, detection probe, and magnesium chloride.

[0012] In one embodiment, the detection probe is labeled with a fluorescent reporter group and a fluorescent quencher group at both ends.

[0013] A recombinase helicase-mediated isothermal amplification method includes the following steps:

[0014] Use the DNA or RNA of the sample to be tested as a template; perform amplification using the enzyme combination product or the kit described above.

[0015] In one embodiment, the amplification reaction system comprises the following components:

[0016]

[0017] In one embodiment, the amplification temperature is 20°C to 42°C, and the amplification time is 5 min to 40 min.

[0018] Compared with traditional technologies, this application has the following advantages:

[0019] This application utilizes molecular biology and bioinformatics methods to screen recombinant protein RecA and helicase UvrD for amino acid mutations. The screened recombinant protein RecAm and helicase UvrDm, when used in isothermal nucleic acid amplification reactions, exhibit increased affinity for DNA, decreased sensitivity to pH, and reduced Mg2+. 2+ Concentration dependence.

[0020] Furthermore, the recombinase helicase-mediated isothermal amplification method for nucleic acids proposed in this application exhibits high specificity, sensitivity, and stability for the target sequence; it also boasts low detection cost and convenient operation, making it promising for widespread application. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The above are fluorescence amplification curves of different RecA mutant recombinant proteins used in Example 1.

[0023] Figure 2 Here is a three-dimensional structural diagram of the recombinant protein RecAm screened in Example 1;

[0024] Figure 3 The graphs show the fluorescence amplification curves of wild-type and mutant recombinant protein RecA used in Example 1 under different pH conditions.

[0025] Figure 4 The wild-type and mutant of recombinant protein RecA used in Example 1 were compared with different Mg... 2+ Fluorescence amplification curves under different concentration conditions;

[0026] Figure 5 This is a fluorescence amplification curve of wild-type and mutant recombinant protein RecA against different concentrations of plasmid template in Example 1;

[0027] Figure 6 This is a three-dimensional structural diagram of the helicase UvrDm screened in Example 2;

[0028] Figure 7 The graphs show the fluorescence amplification curves of wild-type and mutant helicase UvrD used in Example 2 under different pH conditions.

[0029] Figure 8 The following are fluorescence curves of nucleic acid amplification using recombinant protein RecAm and helicase UvrDm under different temperature conditions in Example 3;

[0030] Figure 9 This is a fluorescence curve of the multiplex amplification of different pathogens using recombinant protein RecAm and helicase UvrDm in Example 4;

[0031] Figure 10 The image shows the fluorescence amplification curves for detecting parainfluenza virus using existing kits and the kit described in this application, as shown in Example 5. Detailed Implementation

[0032] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0034] One aspect of this application provides a recombinase combination product for isothermal amplification reactions of nucleic acids.

[0035] In some embodiments, the above-mentioned recombinase combination product includes recombinant protein RecAm and helicase UvrDm; wherein, recombinant protein RecAm has the amino acid sequence shown in SEQ ID NO.2, and helicase UvrDm has the amino acid sequence shown in SEQ ID NO.4.

[0036] RecA protein is a DNA-dependent ATPase that plays a role in bacterial recombinant DNA repair. Its activity is automatically regulated by its C-terminus. Single-stranded DNA-binding protein SSB also indirectly affects RecA function by competing for ssDNA binding sites. RecO, RecR, and possibly RecF proteins all help to load RecA onto SSB-coated ssDNA. UvrD helicase can remove RecA filaments from RecA.

[0037] Single-stranded DNA binding proteins can significantly stimulate the activity of RecA protein in catalyzing homologous pairing of single-stranded DNA, and UvrD helicase can enhance the single-stranded DNA-dependent ATPase activity of RecA by strengthening its affinity for ssDNA. The binding of RecA protein to ATP leads to a significant increase in DNA binding affinity, and ATP binding further stabilizes the RecA-ssDNA structure. Single-stranded DNA binding proteins can form stable SSB-ssDNA complexes with single-stranded ssDNA, but under the influence of RecO, the stability of the SSB-ssDNA complex structure is significantly weakened, while ssDNA is more likely to form RecA-ssDNA complexes. David A. Zarling et al. utilized this characteristic to amplify specific segments of nucleic acid using E. coli RecA protein, SSB protein, and specific primers. Under the influence of ATP, the RecA-primer complex exhibits homologous pairing with complementary sites on the template strand, displacing the strand with the same base sequence. It then binds to the complementary strand to form a D-loop structure. The exposed 3' end of the primer in the D-loop structure is extended by DNA polymerase until replication is complete. This process is repeated cyclically to achieve exponential amplification of the target DNA fragment.

[0038] The recombinant protein RecAm described above can be used as an alternative to recombinases RecA or Rad51, and helicase UvrDm can enhance the chain displacement activity of recombinant protein RecAm.

[0039] In some embodiments, the nucleotide sequence encoding the recombinant protein RecAm is shown in SEQ ID NO.1; and the nucleotide sequence encoding the helicase UvrDm is shown in SEQ ID NO.3.

[0040] In some embodiments, the recombinant protein RecAm is derived from Bacillus subtilis, and the helicase UvrDm is derived from Escherichia coli. By deleting the C-terminal 25 amino acids of the RecA protein from wild-type Bacillus subtilis, the affinity of the RecA protein for DNA is enhanced; simultaneously, the pH tolerance of the RecAm recombinant protein is improved, and the Mg2+ tolerance of the RecAm recombinant protein is reduced. 2+ Dependence. By deleting 40 amino acid residues at the C-terminus of the wild-type Escherichia coli UvrD and site-directedly mutating proline at position 258 of the conserved motif in the middle to leucine, its pH tolerance and stability were enhanced.

[0041] In some embodiments, the recombinant protein RecAm and the helicase UvrDm can be expressed in large quantities in Escherichia coli.

[0042] The recombinase combination product provided in this application includes steps S10 to S40, which are used to prepare the recombinant protein RecAm and the helicase UvrDm.

[0043] S10: Introduce the target gene fragment into the expression vector to construct a recombinant expression plasmid;

[0044] S20: Transform the above recombinant expression plasmid into the expression bacteria to prepare recombinant engineered bacteria;

[0045] S30: The recombinant engineered bacteria prepared in step S20 are induced to express the protein. After enrichment, sonication, and centrifugation of the engineered bacteria, unpurified recombinant protein is prepared.

[0046] S40: The unpurified recombinant protein was purified by chromatography to obtain the recombinant protein RecAm and the helicase UvrDm.

[0047] In some specific examples, in step S10, the target gene expression fragment contains a nucleic acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.3; and the 5' end of the target gene expression fragment has a sticky end with a BamHI restriction site, and the 3' end has a sticky end with a SalI restriction site.

[0048] The purified recombinant protein RecAm exhibits higher affinity for DNA, maintains high activity at higher pH conditions, and remains active at lower Mg2+ levels. 2+ It can also exert its recombinase activity under certain conditions; the purified helicase UvrDm has higher stability under higher pH conditions.

[0049] In the above-mentioned methods for preparing recombinant protein RecAm and helicase UvrDm, in step S10, the expression vector is preferably pET-28a vector.

[0050] In the above-mentioned methods for preparing recombinant protein RecAm and helicase UvrDm, in step S20, the expression bacteria is preferably Escherichia coli.

[0051] The above-mentioned methods for preparing recombinant protein RecAm and helicase UvrDm have the advantages of simple preparation process, high yield and stability, and low mass production cost.

[0052] In some embodiments, the above-described recombinase combination product further includes one or more of single-stranded DNA-binding proteins, BSu DNA polymerase, and creatine phosphokinase.

[0053] This application utilizes molecular biology and bioinformatics methods to screen for amino acid mutations in RecA protein and the helicase UvrD, thereby improving the affinity of RecAm recombinant protein for DNA, reducing its pH sensitivity, enabling it to maintain high recombinase activity even at higher pH conditions, and reducing the Mg2+ oxidase activity of RecAm recombinant protein. 2+ Concentration dependence, which makes it suitable for lower concentrations of Mg 2+ It can also be efficiently amplified under certain environmental conditions. The recombinase combination product of this application includes recombinase RecAm and helicase UvrDm, which can better replace RecA recombinant protein in the RAA reaction; the recombinant protein RecAm has 59% (208 / 353) sequence homology with the wild-type recombinant protein RecA; moreover, the preparation process of recombinase RecAm and helicase UvrDm in this application is simple, the yield and stability are greatly improved, and the mass production cost is low.

[0054] Another aspect of this application provides a kit for isothermal amplification of nucleic acids, the kit comprising the above-described recombinase combination product.

[0055] In some embodiments, the kit may also include one or more of the following: Tris-HCl buffer, potassium chloride, polyethylene glycol 35000, dithiothreitol (DTT), dNTPs, ATP, creatine phosphate, forward primer, directional primer, detection probe, and magnesium chloride.

[0056] In some implementations, the lengths of both the forward and reverse primers are 20 bp to 35 bp.

[0057] In some embodiments, the two ends of the detection probe are labeled with a fluorescent reporter group and a fluorescent quencher group, respectively.

[0058] Another aspect of this application provides a method for recombinase helicase-mediated isothermal amplification of nucleic acids, characterized by comprising the following steps:

[0059] Using the DNA or RNA of the sample to be tested as a template, amplification is performed using the recombinase combination product according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 7.

[0060] In some embodiments, the amplification reaction system includes the following components: Tris-HCl buffer 5mM–100mM, potassium chloride 10mM–150mM, magnesium chloride 1.5mM–15mM, polyethylene glycol 35000 (1%–10% by mass), dithiothreitol 0.5mM–3mM, ATP 1mM–5mM, creatine phosphate 10mM–100mM, dNTPs 0.1mM–1mM, primers 0.2μM–1.0μM, detection probe 0.1μM–0.5μM, creatine phosphate kinase 10ng / μL–100ng / μL, recombinant protein RecAm 10ng / μL–360ng / μL, helicase UvrDm ng / μL–100ng / μL, single-stranded DNA binding protein SSB 200ng / μL–1000ng / μL, Exo III exonuclease 0.5 U / μL~5 U / μL, Bsu DNA polymerase 15 ng / μL~240 ng / μL.

[0061] In some embodiments, the amplification reaction is carried out at a temperature of 20°C to 42°C for a reaction time of 5 min to 40 min.

[0062] In traditional recombinase-mediated strand substitution amplification (RAA) technology, the RecA recombinant protein from *E. coli*, yeast Rad51 recombinase, single-stranded DNA binding protein SSB, and strand substitution DNA polymerase are primarily relied upon. The RecA recombinant protein binds to primers using ATP, forming a RecA recombinant protein-primer complex. This complex recognizes complementary target double-stranded DNA. The recombinant protein-primer complex then invades the 5' end of the target double-stranded DNA, forming a D-loop and decomposing the substrate ATP to produce ADP+Pi. Simultaneously, the strand substitution DNA polymerase binds to the 3' end of the primer for strand extension, forming a new complementary strand. In recombinase-mediated strand substitution amplification, the RecA recombinant protein can alter the reversible process of dissociation and recombination of the RecA-primer complex, directing the reaction in a direction more favorable for amplification.

[0063] Using the recombinant protein RecAm and helicase UvrDm of this application, nucleic acid amplification can be achieved under isothermal conditions, and the amplification reaction has the advantages of being sensitive, efficient, economical, and convenient.

[0064] The recombinase helicase-mediated isothermal amplification (RHMA) method described in this application differs from RAA technology, which only utilizes recombinase activity to achieve strand substitution. RHMA technology, in addition to utilizing the recombinase activity, also utilizes helicase to enhance the affinity between the recombinase and ssDNA, thereby increasing the single-stranded DNA-dependent ATPase activity of the recombinase. This enables rapid amplification of the target nucleic acid, exhibiting strong affinity, high specificity, and high sensitivity for the detection of the target nucleic acid sequence. It can achieve highly sensitive and rapid molecular detection under isothermal conditions of 20℃ to 42℃, with low detection cost, convenient and quick operation, and broad application prospects.

[0065] The reaction principle of the recombinase helicase-mediated isothermal amplification (RHMA) reaction system provided in this application is as follows: (1) The recombinase-ATP-primer complex formed by the recombinase, ATP, and specific primers in the reaction system searches for homologous sequences in the double-stranded DNA template; (2) After the recombinase-ATP-primer complex recognizes the homologous sequence, it initiates a strand exchange reaction, and the single-stranded DNA binding protein then binds to the replaced DNA strand to form a D-Loop structure; (3) The conformation of ATP in the hydrolysis system of the recombinase-ATP-primer complex changes, the recombinase dissociates and exposes the 3' end of the primer, and is recognized by DNA polymerase, which initiates DNA synthesis; (4) DNA polymerase has a strand exchange function, and continues to unwind the double-stranded DNA structure of the template while the primer is extended, and the DNA synthesis process continues; (5) When the two primers are amplified, a complete amplicon is formed; (6) In the reaction system, ATP is hydrolyzed to provide energy for the recombinase and becomes ADP. Under the catalysis of creatine kinase, phosphocreatine can transfer its phosphate group to the ADP molecule to form ATP, thereby restoring the ATP level in the reaction system. Helicase plays a role in enhancing the activity of recombinase and accelerating the reaction process throughout the entire process.

[0066] The above-described RHMA reaction has at least the following advantages:

[0067] (1) In the RHMA reaction system, RecAm was genetically engineered to improve the affinity of RecAm recombinant protein for DNA, which can better recognize low-abundance templates, and has high sensitivity and strong stability.

[0068] (2) During the amplification process, the helicase UvrDm can enhance the activity of recombinant protein RecAm, accelerate the reaction rate, and reduce the reaction time;

[0069] (3) The RHMA reaction system is amplified under constant temperature conditions of 20℃~42℃, which does not require the thermal cycling equipment of PCR technology, thus enabling portable nucleic acid detection, making the detection cost low, the operation convenient and fast, and having broad application prospects.

[0070] (5) The RHMA reaction system can achieve multiplex amplification. Only by adding specific primer pairs to the reaction system, multiple target genes can be detected at once.

[0071] (6) The RHMA reaction system can be used for both DNA template amplification and RNA template amplification.

[0072] (7) RecAm in the RHMA reaction system has a high tolerance pH range. Even if the pH value of the system changes during the lyophilization process, the lyophilized reagent can still maintain high stability and can be stored at room temperature for a long time.

[0073] The present application will be further described below with reference to specific embodiments and comparative examples, but should not be construed as limiting the scope of protection of the present application.

[0074] Example 1: Genetic engineering, purification, preparation, and validation of recombinant protein RecA mutant

[0075] The previously reported RecA recombinant protein exhibits poor activity, weak affinity for DNA, and slow ATP degradation, resulting in low detection sensitivity and long reaction times in isothermal amplification reactions. To address this issue, the applicant has genetically engineered the RecA gene sequence of Bacillus subtilis published in databases, deleting amino acid residues at its C-terminus. The aim is to screen and validate a RecA protein with high DNA affinity and greater tolerance to higher pH and Mg2+ conditions. 2+ RecAm, a recombinant protein with low dependence.

[0076] The applicant genetically engineered the amino acid residues of the primary structural sequence of the activity regulatory site at the C-terminus of the RecA recombinant protein. Deletion mutations of Δ6, Δ13, Δ15, Δ17, and Δ25 amino acid residues were performed at the C-terminus of the RecA recombinant protein, resulting in five mutants.

[0077] (1) Add a sticky end sequence of BamHI restriction site to the 5' end of each of the five mutant target gene fragments and a sticky end sequence of Sall restriction site to the 3' end of each of the five mutant target gene fragments. Then, use gene recombination technology to introduce the five mutant target gene fragments into the pET-28a vector to construct a recombinant expression vector.

[0078] (2) Five recombinant expression vectors were transferred into Escherichia coli by heat shock at 42℃. After a series of processes such as plate coating and screening, recombinant engineered bacteria containing the target gene were obtained.

[0079] (3) The recombinant engineered bacteria were induced and cultured, and the recombinant protein RecAm was obtained after purification. The induction expression method was as follows: when the colony OD value of the recombinant engineered bacteria was 0.4-0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.1 mM / L, and expression was induced at 16℃ for 28 h. The precipitate was collected by solid-liquid separation to obtain the expression bacteria. The engineered expression bacteria were then pulverized by ultrasonication, and the supernatant was collected by solid-liquid separation. The supernatant was subjected to affinity chromatography and then purified by anion exchange chromatography to obtain the target recombinant protein.

[0080] (4) After obtaining the recombinant protein, its affinity for DNA was tested under different pH conditions and Mg. 2+ Enzyme activity at different concentrations. This example uses a recombinant plasmid containing the novel coronavirus 2019nCoV-N gene, and the plasmid at a concentration of 1 ng / μL was diluted 10-fold to 10⁻⁶. -6The concentration was ng / μL, and the materials were prepared as shown in Table 1. The reaction was carried out on a real-time PCR instrument at a reaction temperature of 37℃ for 40 min. Fluorescence was collected every 30 seconds.

[0081] Table 1 Reaction System

[0082] Tris-HCl buffer (pH 8.0) 8mM Potassium chloride 90mM Magnesium chloride 10mM Polyethylene glycol 35000 2.5wt% DTT (dithiothreitol) 1.2mM ATP 2.5mM creatine phosphate 50mM dNTPs 0.2mM forward primer 0.48μM reverse primer 0.48μM creatine phosphokinase 15ng / μL Recombinant protein RecAm 240ng / μL Single-stranded DNA binding protein SSB 500ng / μL Bsu DNA polymerase 120ng / μL SYBR Green I 1× template 2μL Nuclease-Free Water to 25μL

[0083] The primers in this embodiment were designed by the applicant and synthesized by Shanghai Sangon Biotech. The primer sequences for detecting the 2019nCoV-N gene are shown in Table 2.

[0084] Table 2 Primer sequences

[0085] SEQ ID NO.5 SARS-NF GAACTTCTCCTGCTAGAATGGCTGGCAATGGCG SEQ ID NO.6 SARS-NR GCAGATTTCTTAGTGACAGTTTGGCCTTGTTGT

[0086] (5) Affinity verification of different mutants: Affinity was verified using the mutant recombinant proteins RecA△6C, RecA△13C, RecA△15C, RecA△17C, RecA△25C, and wild-type recombinant protein RecA (NEB: M0249S) according to the reaction systems prepared in Table 1. The results are as follows: Figure 1 As shown: Compared to the wild-type E. coli RecA recombinant protein, among the five mutants obtained through genetic engineering in this embodiment, RecA△6C, RecA△13C, and RecA△15C showed lower affinity for low-abundance DNA templates than the wild-type; while the two mutants RecA△17C and RecA△25C showed significantly higher affinity for low-abundance DNA templates than the wild-type, with the RecA△25C mutant showing a higher affinity for low-abundance DNA templates than the RecA△17C mutant; therefore, RecA△25C was named the recombinant protein RecAm; the three-dimensional structure of this recombinant protein RecAm is shown below. Figure 2 As shown.

[0087] The gene sequence of the recombinant protein RecAm is shown in SEQ ID NO.As shown in 1: ATGAGTGATCGTCAGGCAGCCTTAGATATGGCTCTTAAACAAATAGAAAAACAGTTCGGCAAAGGTTCCATTATGAAACTGGGAGAAAAGACAGATACAAGAATTTCTACTGTACCAAGCGGCTCCCTCGCTCTTGATACAGCACTGGGAATTGGCGGATATCCTCGCGGACGGATTATTGAAGTATACGGTCCTGAAAGCTCAGGTAAAACAACTGTGGCGCTTCATGCGATTGCTGAAGTTCAGCAGCAGGGCGGACAAGCCGCGTTTATCGATGCGGAGCATGCGTTAGATCCGGTATACGCGCAAAAGCTCGGTGTTAACATCGAAGAGCTTTTACTGTCTCAGCCTGACACAGGCGAGCAGGCGCTTGAAATTGCGGAAGCATTGGTTCGAAGCGGGGCAGTTGACATTGTCGTTGTCGACTCTGTAGCCGCTCTCGTTCCGAAAGCGGAAATTGAAGGCGACATGGGAGATTCGCATGTCGGTTTACAAGCACGCTTAATGTCTCAAGCGCTTCGTAAGCTTTCAGGGGCCATTAACAAATCGAAGACAATCGCGATTTTCATTAACCAAATTCGTGAAAAAGTCGGTGTTATGTTCGGGAACCCGGAAACAACACCTGGCGGCCGTGCGTTGAAATTCTATTCTTCCGTGCGTCTTGAAGTGCGCCGTGCTGAACAGCTGAAACAAGGCAACGACGTAATGGGGAACAAAACGAAAATCAAAGTCGTGAAAAACAAGGTGGCTCCGCCGTTCCGTACAGCCGAGGTTGACATTATGTACGGAGAAGGCATTTCAAAAGAAGGCGAAATCATTGATCTAGGAACTGAACTTGATATCGTGCAAAAAAGCGGTTCATGGTACTCTTATGAAGAAGAGCGTCTTGGCCAAGGCCGTGAAAATGCAAAACAATTCCTGAAAGAAAATAAAGATATCATGCTGATGATCCAGGAGCAAATTCGCGAATAA.

[0088] The amino acid sequence of recombinant protein RecAm is as SEQ ID Shown in NO.2: MSDRQAALDMALKQIEKQFGKGSIMKLGEKTDTRISTVPSGSLALDTALGIGGYPRGRIIEVYGPESSGKTTVALHAIAEVQQQGGQAAFIDAEHALDPVYAQKLGVNIEELLLSQPDTGEQALEIAEALVRSGAVDIVVVDSVAALVPKAEIEGDMG DSHVGLQARLMSQALRKLSGAINKSKTIAIFINQIREKVGVMFGNPETTPGGRALKFYSSVRLEVRRAEQLKQGNDVMGNKTKIKVVKNKVAPPFRTAEVDIMYGEGISKEGEIIDLGTELDIVQKSGSWYSYEEERLGQGRENAKQFLKENKDIMLMIQEQIRE

[0089] (6) Enzyme activity verification of mutant recombinant enzymes under different pH conditions: Tris-HCl buffer solutions with pH values ​​of 8.0, 8.4, 8.6, 8.8, and 9.0 were prepared. Other components are shown in Table 1. Enzyme activity verification was performed on mutant recombinant proteins RecAΔ17C, ​​RecAΔ25C, and wild-type recombinant protein RecA. Results are shown below. Figure 3 As shown, the wild-type Escherichia coli RecA recombinant protein exhibits high recombinase activity at pH 8.0, while the two mutants, RecA△17C and RecA△25C, still exhibit high recombinase activity at pH 9.0.

[0090] (7) Mutant recombinant enzymes in different Mg 2+ Enzyme activity verification under concentration conditions: Preparation of Mg 2+ Reagents at concentrations of 2.5 mM, 5 mM, and 10 mM, with other components listed in Table 1, were used to verify the enzyme activities of the mutant recombinant protein RecAΔ17C, ​​RecAΔ25C, and the wild-type recombinant protein RecA. The results are as follows: Figure 4 As shown: Wild-type Escherichia coli RecA recombinant protein in 10 mM Mg 2+ The two mutants, RecA△17C and RecA△25C, exhibited high recombinase activity at 2 mM Mg2+ concentrations. 2+ It exhibits high recombinase activity at certain concentrations, showing a high degree of activity against Mg. 2+ Dependence decreases.

[0091] (8) Amplification efficiency and sensitivity analysis of mutant recombinase and wild-type recombinase: According to the reaction system in Table 1, reaction systems were prepared using the recombinant protein RecAm and wild-type recombinant protein RecA screened in this example, respectively. The amplification efficiency and sensitivity analysis were performed using a concentration of 10... -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 Recombinant plasmids containing ng / μL of the 2019nCoV-N gene of the novel coronavirus were amplified using an ABI Q5 real-time PCR instrument at 37℃ for 45 cycles, with fluorescence collected every 30 seconds. Under the same reaction time, the detection Ct values ​​of different recombinant proteins were compared to analyze the amplification efficiency and sensitivity of the recombinase.

[0092] The detection results showed that there was a significant difference in the detection Ct values ​​of recombinant protein RecAm and wild-type recombinant protein RecA for the same concentration of plasmid samples. The detection Ct value of recombinant protein RecAm was significantly smaller than that of wild-type recombinant protein RecA (Table 3), indicating that the amplification efficiency of recombinant protein RecAm was higher than that of wild-type recombinant protein. Furthermore, within the 45 detected Ct values, recombinant protein RecAm showed amplification curves for all detected samples, while wild-type recombinant protein RecA showed amplification curves for 10 Ct values. -6 No amplification curve was observed with ng / μL plasmid. Figure 5 This indicates that the recombinant protein RecAm has a higher sensitivity than the wild-type recombinant protein.

[0093] Table 3 Comparison of Ct detection results between recombinant protein RecAm and wild-type recombinant protein RecA.

[0094] 1 <![CDATA[10 -1 ng / μL]]> 4.588 10.770 2 <![CDATA[10 -2 ng / μL]]> 6.116 12.946 3 <![CDATA[10 -3 ng / μL]]> 8.172 16.896 4 <![CDATA[10 -4 ng / μL]]> 9.478 25.847 5 <![CDATA[10 -5 ng / μL]]> 16.348 37.615 6 <![CDATA[10 -6 ng / μL]]> 27.944 /

[0095] Example 2: Genetic engineering, purification, preparation, and verification of helicase UvrD

[0096] UvrD helicase is an enzyme that uses NTPs to hydrolyze and release nucleic acids, playing a crucial role in DNA replication and manipulation. However, UvrD helicase exhibits lower nucleic acid melting activity in solutions with high salt, high pH, ​​and high glycerol concentrations. The applicant has proposed a mutant UvrD helicase that indirectly enhances the RecAm chain substitution activity of recombinant protein and is more tolerant to higher pH levels by deleting 40 amino acid residues at the C-terminus of the published E. coli UvrD helicase and site-directedly mutating proline at position 258 of the conserved motif in the middle to leucine.

[0097] In this embodiment, the applicant obtained the helicase UvrD mutant gene sequence by deleting 40 amino acid residues at the C-terminus of the published E. coli UvrD in the database and by site-directed mutation of proline at position 258 in the intermediate conserved motif to leucine.

[0098] 1) Add a sticky end sequence of BamHI restriction site to the 5' end and a sticky end sequence of Sall restriction site to the 3' end of the target gene fragment of the helicase mutant. Then, introduce the target gene fragment of the mutant into the pET-28a vector using gene recombination technology to construct a recombinant expression vector.

[0099] 2) The recombinant expression vector was transformed into Escherichia coli by heat shock at 42℃, and the recombinant engineered bacteria containing the target gene were obtained through a series of processes such as plate coating and screening.

[0100] 3) The recombinant engineered bacteria were induced and cultured, and the helicase UvrD was obtained after purification. The induction method was as follows: when the colony OD value of the recombinant engineered bacteria was 0.4-0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.1 mM / L and expression was induced at 16℃ for 28 h. The precipitate was collected by solid-liquid separation to obtain the expression bacteria. The engineered expression bacteria were then ultrasonically pulverized, and the supernatant was collected by solid-liquid separation. The supernatant was subjected to affinity chromatography, followed by anion exchange chromatography purification to obtain the target helicase UvrDm, whose three-dimensional structure is shown in the figure. Figure 6 As shown.

[0101]

[0102] The amino acid sequence of helicase UvrDm is as SEQ ID Shown in NO.4: MDVSYLLDSLNDKQREAVAAPRSNLLVLAGAGSGKTRVLVHRIAWLMSVENCSPYSIMAVTFTNKAAAEMRHRIGQLMGTSQGGMWVGTFHGLAHRLLRAHHMDANLPQDFQILDSEDQLRLLKRLIKAMNLDEKQWPPRQAMWYINSQKDEGLRPHHIQSYGN PVEQTWQKVYQAYQEACDRAGLVDFAELLLRAHELWLNKPHILQHYRERFTNILVDEFQDTNNIQYAWIRLLAGDTGKVMIVGDDDQSIYGWRGAQVENIQRFLNDFPGAETIRLEQNYRSTSNILSAANALIENNNGRLGKKLWTDGADGEPISLYCAFNELDEARFVVNR IKTWQDNGGALAECAILYRSNAQSRVLEEALLQASMPYRIYGGMRFFERQEIKDALSYLRLIANRNDDAAFERVVNTPTRGIGDRTLDVVRQTSRDRQLTLWQACRELLQEKALAGRAASALQRFMELIDALAQETADMPLHVQTDRVIKDSGLRTMYEQEKGEKGQTRIEN LEELVTATRQFSYNEEDEDLMPLQAFLSHAALEAGEGQADTWQDAVQLMTLHSAKGLEFPQVFIVGMEEGMFPSQMSLDEGGRLEEERRLAYVGVTRAMQKLTLTYAETRRLYGKEVYHRPSRFIGELPEECVEEVRLRATVSRPVSHQRMGTPMVENDSGYKLGQRVRHAK

[0103] After obtaining the target helicase UvrDm, the effect of UvrDm on the chain displacement activity of RecAm protein under high pH conditions was investigated using a synergistic approach between UvrDm and RecAm protein in nucleic acid amplification. The recombinant plasmid containing the novel coronavirus 2019nCoV-N gene was diluted 10-fold from 1 ng / μL to 10... -6 The sample was collected at ng / μL using the same primers and configurations as shown in Table 4 in Example 1. The reaction was performed on a real-time PCR instrument at a temperature of 37°C for 40 min, with fluorescence collected every 30 seconds.

[0104] Table 4 Reaction System

[0105] Tris-HCl buffer (pH 8.0) 8mM Potassium chloride 90mM Magnesium chloride 2.5mM Polyethylene glycol 35000 2.5wt% DTT 1.2mM ATP 2.5mM creatine phosphate 50mM dNTPs 0.2mM forward primer 0.48μM reverse primer 0.48μM creatine phosphokinase 15ng / μL Recombinant protein RecAm 240ng / μL helicase UvrDm 40ng / μL Single-stranded DNA binding protein SSB 500ng / μL Bsu DNA polymerase 120ng / μL SYBR Green I 1× template 2μL Nuclease-Free (NF) Water to 25μL

[0106] (4) The effect of higher pH conditions on the chain displacement activity of recombinant protein RecAm was studied, and the results are as follows: Figure 7 As shown: at pH 8.0, the wild-type Escherichia coli helicase UvrD stimulates the recombinant protein RecAm to exhibit high chain substitution activity; while at pH 9.0, the helicase UvrDm of this application stimulates the recombinant protein RecAm to exhibit high chain substitution activity.

[0107] Example 3: Application of Recombinase Helicase-Mediated Isothermal Amplification (RHMA) at Different Temperatures

[0108] Based on the recombinant protein RecAm and helicase UvrDm of this application, a recombinase-mediated isothermal amplification technique was constructed. The recombinant plasmid containing the novel coronavirus 2019nCoV-N gene was diluted 10-fold from 1 ng / μL to 10... -7 The sample was prepared using the same primers (Table 2) and reaction system as in Example 1 and Table 4. The reaction was carried out on a real-time PCR instrument at temperatures of 20℃, 25℃, 30℃, 37℃, and 42℃ for 40 min, with fluorescence collected every 30 sec.

[0109] The results are as follows Figure 8 As shown, the reaction reagent amplified signals for all templates at temperatures of 20℃, 25℃, 30℃, 37℃, and 42℃.

[0110] Example 4: Application of Recombinase Helicase-Mediated Isothermal Amplification (RHMA) in Multiplex Detection of Respiratory Pathogens

[0111] This embodiment uses the detection of SARS-CoV-2, H1N1 influenza A virus, and influenza B virus as examples to illustrate the application of recombinase-mediated isothermal amplification technology. For different microbial pathogens, specific primers only need to be designed based on the pathogen's gene sequence. The primers in this embodiment were designed by the applicant and synthesized by Shanghai Sangon Biotech.

[0112] Specifically, this is achieved through the following steps: The SARS-CoV-2 ORF1ab gene, the MP gene of influenza A virus H1N1, and the MP gene of influenza B virus are selected, BLAST analysis is performed, and specific primers are designed for each gene. The designed primers only perform effective isothermal amplification of their respective genes. Primer information is shown in Table 5 (in the probe sequences, FAM-dT represents thymidine nucleotides carrying fluorescent groups, THF represents the abase analog tetrahydrofuran, BHQ-dT represents thymidine nucleotides carrying fluorescence quenching groups, and C3 Spacer represents the intercalary arm).

[0113] Table 5 Primer Information

[0114]

[0115] In a biosafety cabinet, the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver. 5.0 (Takara: 9766) was used to extract RNA from pseudoviruses containing the complete gene sequence of the novel coronavirus (Shanghai Yisheng: 11900ES03), H1N1 influenza A virus ribonucleic acid (H1N1 RNA) liquid internal quality control (Guangzhou Bangdesheng: BDS-IQC-173), and influenza B virus ribonucleic acid (IVB RNA) liquid internal quality control (Yamagata strain) (Guangzhou Bangdesheng: BDS-IQC-316). After preparing the reaction system according to Table 6, the PCR tubes were capped and placed in a real-time PCR instrument for amplification. The reaction temperature was 42℃, the reaction time was 40 min, and fluorescence was collected every 30 seconds.

[0116] Table 6 Reaction System

[0117]

[0118]

[0119] Multiplex amplification results as follows Figure 9 As shown, except for NTC which did not show a peak, the novel coronavirus, influenza A virus H3N2, and influenza B virus all showed amplification signals, indicating that the reagents in this application can perform multiple strand displacement amplification reactions.

[0120] Example 5: Sensitivity Analysis of Detection Based on Recombinase Helicase-Mediated Isothermal Amplification (RHMA) Technology

[0121] This embodiment uses the detection of parainfluenza virus type 1 HN gene plasmid as an example to specifically illustrate the comparison of analytical sensitivity between recombinase helicase-mediated isothermal amplification (RHMA) and comparative reagents. In this embodiment, the parainfluenza virus type 1 HN gene plasmid was diluted from 1 ng / μL down in a 10-fold concentration gradient to 10... -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 - 4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL, 10 -7 ng / μL was used as a sample for the amplification reaction for comparative analysis.

[0122] Specifically, this is achieved through the following steps: After selecting the HN gene of parainfluenza virus type 1 and performing BLAST alignment analysis, specific primers and exo probes were designed. In this embodiment, the primers were designed by the applicant and synthesized by Shanghai Sangon Biotech; primer information is shown in Table 7.

[0123] Table 7 Primer Information

[0124]

[0125] The DNA was amplified using RT-basic nucleic acid amplification reagent (RAA method) purchased from Hangzhou Zhongce Biotechnology, with the addition of exoIII exonuclease plasmid. The system was prepared according to the system shown in Table 8. After preparation, 48 μL of reconstitution solution was added to each lyophilized reaction tube, and then 2 μL of nucleic acid template was added after reconstitution.

[0126] Table 8 RAA Reaction System

[0127]

[0128]

[0129] The reaction system for the 25 μL recombinase helicase-mediated isothermal amplification technique is shown in Table 9.

[0130] Table 9 RHMA Reaction System

[0131] Tris-HCl buffer (pH 8.0) 10mM Potassium chloride 90mM Magnesium chloride 2.5mM Polyethylene glycol 35000 2.5wt% DTT 1.2mM ATP 2.5mM creatine phosphate 50mM dNTPs 0.2mM forward primer 0.48μM reverse primer 0.48μM probe 0.12μM creatine phosphokinase 15ng / μL Recombinant protein RecAm 240ng / μL helicase UvrDm 40ng / μL Single-stranded DNA binding protein SSB 500ng / μL Bsu DNA polymerase 120ng / μL M-MLV reverse transcriptase 2U / μL RNasin 0.1 U / μL Exo III 2U / μL template 2μL NF-Water to 25μL

[0132] After the reaction system is prepared, the PCR tube is capped and placed on a real-time PCR instrument for amplification. The reaction temperature is 42℃ and the reaction time is 40 min. Fluorescence is collected every 30 seconds.

[0133] Sensitivity analysis results are as follows Figure 10 As shown, the RT-basic nucleic acid amplification reagent (RAA method) from Hangzhou Zhongce manufacturer, when used with exo III exonuclease to amplify plasmid DNA, could only detect 10. -6 plasmid concentration in ng / μL ( Figure 10 (A) but the reagent described in this application can detect 10 -7 Plasmid concentration of ng / μL ( Figure 10 (B) indicates that the reagents in this application have higher detection sensitivity than the RT-basic nucleic acid amplification reagents produced by Hangzhou Zhongce.

[0134] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0135] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. An enzyme combination product for isothermal amplification reactions of nucleic acids, characterized in that, It includes a recombinant protein RecAm and a helicase UvrDm; the amino acid sequence of the recombinant protein RecAm is shown in SEQ ID NO. 2; the nucleotide sequence encoding the helicase UvrDm is shown in SEQ ID NO.

3.

2. The enzyme combination product according to claim 1, characterized in that, The nucleotide sequence encoding the recombinant protein RecAm is shown in SEQ ID NO.

1.

3. The enzyme combination product according to any one of claims 1 to 2, characterized in that, It also includes one or more of single-stranded DNA-binding proteins, Bsu DNA polymerase, and phosphocreatine kinase.

4. A reagent kit for isothermal amplification of nucleic acids, characterized in that, Includes the enzyme combination product according to any one of claims 1 to 3.

5. The reagent kit according to claim 4, characterized in that, It also includes one or more of the following: Tris-HCl buffer, potassium chloride, polyethylene glycol 35000, dithiothreitol, dNTPs, ATP, creatine phosphate, forward primer, reverse primer, detection probe, and magnesium chloride.

6. The reagent kit according to claim 5, characterized in that, The detection probe is labeled with a fluorescent reporter group and a fluorescent quencher group at both ends.

7. A non-diagnostic recombinase helicase-mediated isothermal amplification method, characterized in that, Includes the following steps: Using the DNA or RNA of the sample to be tested as a template, amplification is performed using the enzyme combination product according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 6.

8. The method according to claim 7, characterized in that, The amplification reaction system includes the following components: Tris-HCl buffer 5mM~100mM Potassium chloride 10mM~150mM Magnesium chloride 1.5mM~15mM Polyethylene glycol 35000 1wt%~10wt% Dithiothreitol 0.5mM~3mM ATP 1mM~5mM Creatine phosphate 10mM~100mM dNTPs 0.1mM~1mM Forward primers: 0.2 μM ~ 1.0 μM Reverse primers 0.2 μM~1.0 μM Detection probe 0.1μM~0.5μM Creatine phosphokinase 10 ng / μL ~ 100 ng / μL Recombinant protein RecAm 10 ng / μL ~ 360 ng / μL helicase UvrDm 10ng / μL~100ng / μL Single-stranded DNA binding protein 200 ng / μL~1000 ng / μL Exo III exonuclease 0.5 U / μL~5 U / μL, and Bsu DNA polymerase 15 ng / μL ~ 240 ng / μL.

9. The method according to any one of claims 7 to 8, characterized in that, The amplification temperature is 20℃~42℃, and the amplification time is 5min~40min.

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