CN112592905A - DNA polymerase mixture for novel coronavirus detection - Google Patents

Abstract

The invention provides a DNA polymerase mixture for detecting novel coronavirus, and particularly the invention obtains significantly higher PCR amplification efficiency by mixing wild type DNA polymerase and mutant DNA polymerase mutant 17, and the mixed use of the wild type DNA polymerase and the mutant DNA polymerase mutant 17 shows significant synergistic effect.

Description

DNA polymerase mixture for novel coronavirus detection

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mixture for detecting novel coronavirus.

Background

Taq enzyme is a thermostable DNA polymerase derived from thermostable bacterium Thermus aquaticus, has a molecular weight of 94KDa, has an optimum reaction temperature of 75-80 ℃ in the presence of magnesium ions, has an activity half-life of 40 minutes at 95 ℃, and has 5 '-3' exonuclease activity. Because of its high temperature resistance, it is widely used in Polymerase Chain Reaction (PCR) and is the first enzyme for nucleic acid amplification and detection. The commercialized Taq enzyme is cloned and expressed using an Escherichia coli prokaryotic expression system.

The modern molecular biological detection technology has higher and higher requirements on the sensitivity, the accuracy and the durability of PCR reaction, and the wild Taq enzyme cannot completely meet the requirements of practical application. To make it more adaptable to the use of specific technologies, many attempts have been made to mutate the Taq enzyme sequence, such as:

DNA binding domains were added to confer stronger elongation activity (Wang Y (2004). A novel strand to engineering DNA polymers for enhanced processing and enhanced performance in vitro. nucleic Acids Res 32, 1197-1207));

higher fidelity of the DNA by site-directed mutagenesis and deletion of the domain (Suzuki M, Yoshida S, Adman ET, Blank A, Loeb LA (2000) Thermus Aquaticus DNA polymerase I mutations with altered fidelity. interactions in the O-Helix. J Biol Chem275:32728 32735), higher DNA polymerization activity (Mutant Taq DNA polymerase with improved elasticity as a used expression reagent for genetic engineering. Front Microbiol 5:461.doi: 10.3389/fmic. 2014.00461), high concentration inhibitors (Zhang Z, Kermekchiev MB, Barnes WM (2010) Direct DNA amplification from clinical samples using a PCR enhancer cocktail and novel variants of Taq. J Mol Diagn 12: 152-161), reduction of 5 '-3' exonuclease activity (Vainshtein I, Atrazhev A, Eom SH, Elliott JF, Wishart DS, Malcolm BA (1996) Peptide restriction of an N-Terminal cleavage of the storage fragment of Taq DNA polymerase. protein Sci 5: 51785-51792).

Those skilled in the art have worked to develop DNA polymerases with higher amplification efficiencies to meet the needs of modern molecular biological detection techniques.

Disclosure of Invention

The invention aims to provide a DNA polymerase mixture for detecting novel coronavirus.

In a first aspect of the invention, there is provided a DNA polymerase mixture comprising a wild-type DNA polymerase and a mutant DNA polymerase; wherein the amino acid sequence of the wild type DNA polymerase is shown as SEQ ID NO. 2;

the mutant DNA polymerase is selected from the group consisting of:

mutant 1: mutation is performed on the basis of the wild-type DNA polymerase shown in SEQ ID No. 2, and the mutant 1 has the following mutation sites: E507A, K508L, E734E, F749K;

mutant 6: mutation is performed on the basis of the wild-type DNA polymerase shown in SEQ ID No. 2, and the mutant 6 has the following mutation sites: H785G;

mutant 17: the mutation is performed on the basis of the wild-type DNA polymerase shown in SEQ ID No. 2 and the mutant 17 has the following mutation sites: E734F, Y672P.

In another preferred embodiment, the mutation sites of the mutant 1 are: E507A, K508L, E734E, F749K.

In another preferred embodiment, the mutation sites of mutant 6 are: H785G.

In another preferred embodiment, the mutation sites of mutant 17 are: E734F, Y672P.

In another preferred embodiment, the enzyme activity ratio of the wild-type DNA polymerase to the mutant-type DNA polymerase in the DNA polymerase mixture is about 1-9: 9: 1; preferably about 2 to about 8: 8: 2; more preferably about 3 to about 7: 7: 3.

in a second aspect of the invention, there is provided a kit comprising a DNA polymerase mixture according to the first aspect of the invention.

In another preferred embodiment, the kit further comprises a PCR buffer.

In another preferred embodiment, the kit further comprises a reverse transcriptase, and/or dntps.

In a third aspect of the present invention, there is provided a PCR amplification method, comprising the steps of:

(1) providing a nucleic acid sample of an object to be detected;

(2) preparing a PCR reaction system and carrying out PCR reaction:

wherein in step (2), the DNA polymerase mixture of the first aspect of the invention is used to catalyze the polymerization of substrate dNTP molecules to form progeny DNA.

In another preferred embodiment, the nucleic acid sample comprises cDNA of the novel coronavirus SARS-CoV-2.

In another preferred embodiment, the method is a method for non-diagnostic purposes, such as the detection of environmental samples or laboratory animal or cultured cell samples.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found that a mixture of a wild-type DNA polymerase and a specific mutant DNA polymerase has a higher PCR amplification efficiency, and the wild-type DNA polymerase and the specific mutant DNA polymerase exhibit a significant synergistic effect. On the basis of this, the present invention has been completed.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, 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 invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Taq enzyme

Taq enzyme is widely used in Polymerase Chain Reaction (PCR) and is the first enzyme for nucleic acid amplification, detection and other reactions. The commercialized Taq enzyme is cloned and expressed using an Escherichia coli prokaryotic expression system.

The DNA sequence of the wild type Taq enzyme is as follows:

ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAA(SEQ ID NO.:1)

the amino acid sequence of the wild Taq enzyme is as follows:

MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE(SEQ ID NO.:2)

the Taq enzyme gene sequence of the present invention can be obtained by a conventional method used by those skilled in the art, for example, a total artificial synthesis or a PCR synthesis. One preferred synthesis method is the asymmetric PCR method. The asymmetric PCR method uses a pair of primers with different amounts to generate a large amount of single-stranded DNA (ssDNA) after PCR amplification. The pair of primers are referred to as non-limiting and limiting primers, respectively, and are typically in a ratio of 50-100: 1. In the first 10-15 cycles of the PCR reaction, the amplification product is mainly double-stranded DNA, but when the restriction primers (low concentration primers) are consumed, PCR using non-restriction primers (high concentration primers) will produce a large amount of single-stranded DNA. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The Taq enzyme of the present invention can be expressed or produced by a conventional recombinant DNA technique, comprising the steps of:

(1) transforming or transducing a suitable host cell with a polynucleotide encoding a protein of the invention, or with a recombinant expression vector containing the polynucleotide;

(2) culturing the host cell in a suitable medium;

(3) separating and purifying the target protein from the culture medium or the cells to obtain the Taq enzyme.

Methods well known to those skilled in the art can be used to construct expression vectors comprising the DNA sequences encoding the Taq enzyme of the invention and suitable transcription/translation control signals, preferably commercially available vectors: pET 28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

The recombinant vector comprises in the 5 'to 3' direction: a promoter, a gene of interest, and a terminator. If desired, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polyadenylation signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; selection markers (antibiotic resistance genes, fluorescent proteins, etc.); an enhancer; or operator.

Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In general, any plasmid and vector may be used as long as it can replicate and is stable in the host.

One of ordinary skill in the art can construct vectors containing the promoter and/or gene sequence of interest of the present invention using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The expression vector of the present invention can be used to transform an appropriate host cell so that the host transcribes the target RNA or expresses the target protein. The host cell may be a prokaryotic cell, such as E.coli, C.glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., Escherichia coli), CaCl may be used₂The treatment can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may be transformed by methods such as Agrobacterium transformation or biolistic transformation, for example, leaf disc method, immature embryo transformation, flower bud soaking method, etc. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain transgenic plants.

The term "operably linked" means that the gene of interest to be expressed transcriptionally is linked to its control sequences in a manner conventional in the art to be expressed.

Culture of engineering bacteria and fermentation production of target protein

After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the invention. The medium used in the culture may be selected from various conventional media, depending on the host cell, and the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

In the present invention, conventional fermentation conditions may be employed. Representative conditions include (but are not limited to):

(a) the temperature is kept between 25 and 37 ℃ for the fermentation and induction of the Taq enzyme;

(b) the pH value of the induction phase is controlled to be 3-9;

(c) in the case of Dissolved Oxygen (DO), DO is controlled at 10-90%, and the maintenance of dissolved oxygen can be solved by introducing oxygen/air mixed gas;

(d) for feeding, the feeding type preferably comprises carbon sources such as glycerol, methanol, glucose and the like, and the feeding can be carried out independently or in a mixed manner;

(e) as for the IPTG concentration during induction, conventional induction concentrations can be used in the present invention, and usually the IPTG concentration is controlled at 0.1-1.5 mM;

(f) the induction time is not particularly limited, and is usually 2 to 20 hours, preferably 5 to 15 hours.

The Taq enzyme, a target protein, exists in cells of escherichia coli, host cells are collected through a centrifugal machine, then the host cells are crushed through high pressure, mechanical force, enzymatic cell disruption or other cell crushing methods, and recombinant proteins are released, wherein the high pressure method is preferred. The host cell lysate can be primarily purified by flocculation, salting out, ultrafiltration, etc., and then purified by chromatography, ultrafiltration, etc., or directly purified by chromatography.

The chromatography includes cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. Common chromatographic methods include:

1. anion exchange chromatography:

anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is higher, affecting binding to the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography is performed. The sample can be replaced by means of dilution, ultrafiltration, dialysis, gel filtration chromatography and the like until the sample is similar to a corresponding ion exchange column equilibrium liquid system, and then the sample is loaded and subjected to gradient elution of salt concentration or pH.

2. Hydrophobic chromatography:

hydrophobic chromatographic media include (but are not limited to): Phenyl-Sepharose, Butyl-Sepharose, octyl-Sepharose. Samples were prepared by adding NaCl, (NH)₄)₂SO₄And increasing the salt concentration, loading, and eluting by decreasing the salt concentration. The hetero-proteins having large differences in hydrophobicity were removed by hydrophobic chromatography.

3. Gel filtration chromatography

Hydrophobic chromatographic media include (but are not limited to): sephacryl, Superdex, Sephadex types. The buffer system is replaced by gel filtration chromatography or further purified.

4. Affinity chromatography

Affinity chromatography media include (but are not limited to): HiTrap^TMHeparinHPColumns。

5. Membrane filtration

The ultrafiltration medium comprises: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, metal membranes. The purification and concentration can be achieved by membrane filtration.

The main advantages of the invention are:

the DNA polymerase mixture of the invention has higher PCR amplification efficiency, thus being capable of improving the detection efficiency and the detection accuracy of the novel coronavirus.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

The preparation method and the preparation method of the Taq enzyme mutant in the patent document CN201910083410.X are cited in the embodiment of the invention and are as follows.

Example 1: construction of random mutation plasmid of Taq enzyme

Amplifying a polymerase activity structural domain DNA sequence (423-831 amino acid coding sequence) of the Taq enzyme by using low-fidelity PCR (Error-PCR), wherein the mutation occurrence rate is 0.3 percent, then connecting with the rest coding sequences (1-423 amino acid sequence) of the Taq enzyme, and cloning into a pET28a prokaryotic expression vector to obtain the Taq enzyme random mutation plasmid. The method comprises the following specific steps:

1) using Taq-pET28a plasmid as template, designing primer T (1-423) to amplify Taq (1-423).

Taq(1-423)DNA Seq

ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAG(SEQ ID NO.:3)

T1-423_PF：5'ATATCATATGCGTGGCATGCTGCCGCTTTT 3'(SEQ ID NO.:4)

T1-423_PR：5'GCATGAATTCCGTCTCCTCTCCCTCTAAGC 3'(SEQ ID NO.:5)

PCR reaction system and procedure:

PCR procedure: 95 ℃ for 3 minutes, (95 ℃ for 30 seconds, 60 ℃ for 30 seconds, 72 ℃ for 1 minute) x25 cycles, 72 ℃ for 3 minutes, 4 ℃ storage

The PCR product was purified and recovered with a DNA gel recovery kit, digested with NdeI and XhoI, ligated to pET28a vector, and sequenced to confirm that the sequence was correct, and the resulting plasmid was named Taq (1-423) -pET28

2) Using Clontech and Taq-pET28a plasmid as a template

PCR Random Mutagenesis Kit (Dalianbao organism PT3393-2), designing primer (TMu _ F/R) to amplify Taq (423-

Taq(423-832)DNA Seq

GGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAATAA(SEQ ID NO.:6)

TMu_F：5'GGAGAGGAGCGCCTGTTGTGGTTGT 3'(SEQ ID NO.:7)

TMu_R：5'TTATTCCTTCGCAGATAACCAGTCT 3'(SEQ ID NO.:8)

PCR reaction system and procedure:

3 min at 95 deg.C, (30 sec at 95 deg.C, 30 sec at 60 deg.C, 2 min at 68 deg.C) X25 cycles, 5 min at 68 deg.C, and 4 deg.C

The PCR product is cut by BsmBI and XhoI enzyme, then Taq (1-423) -pET28 plasmid cut by BsmBI and XhoI enzyme is connected, the connection product is transformed into BL21(DE3) expression host bacteria, and the number of transformants is counted.

Example 2: expression and directed evolution screening of Taq enzyme mutants

The Taq enzyme mutation plasmid is transformed into a BL21(DE3) expression strain, and a Taq enzyme mutation library is induced and expressed. BL21(DE3) induced expression bacteria containing Taq enzyme mutation library are dispersed and wrapped by an emulsion PCR system, PCR reaction is carried out, and DNA containing Taq enzyme mutation fragments is amplified. Then, the DNA fragment amplified by the emulsion PCR is subjected to high fidelity PCR secondary amplification by using a Taq enzyme specific primer, and the amplified DNA product is cloned into a pET28a expression vector again to complete a screening process. Then, the screening process of emulsion PCR-secondary high-fidelity PCR-cloning to pET28a expression vector is repeated, and the extension time of emulsion PCR in each screening is gradually shortened, so that the mutant population with high extension activity and high amplification activity is accumulated. The method comprises the following specific steps:

1) the transformant obtained by transformation in example 1 was inoculated into LB medium, shake-cultured at 37 ℃ for 6 hours, added with isopropyl thiogalactoside (IPTG) at a final concentration of 0.1mM, and induced at 37 ℃ for 3 hours. The cells were collected by centrifugation and treated with ddH₂O washing the cells twice, and finallyBy ddH₂O resuspending the cells, measuring the light absorption at 600nm (OD 600) of the cell solution, and diluting with ddH2O to a final concentration of 1.0 OD600

2) Preparing oil phase solution

Tween-80 200ul

Triton X-100 25ul

Mineral oil 10ml

Mixing the above 3 reagents, and mixing

3) Preparation of aqueous reaction solution

The cell suspension prepared in step 1) and having an OD600 of 1.0 was diluted 100-fold with ddH2O to prepare the following reaction solution

10XTaq enzyme reaction (100mM Tris, 500mM KCl, 100mM (NH4)2SO4, pH8.0)13ul

pET28_ F primer: TACGGTTAACCCTTTGAATCA (SEQ ID NO. 9)

pET28_ R primer: GTTACCTGGTTAAACTGTACT (SEQ ID NO. 10)

4) Preparation of the emulsion System

Mixing 200ul of water phase and 400ul of oil phase in a 2ml tube, oscillating for 10 minutes at high speed on a vortex oscillator, taking 5 PCR tubes, subpackaging 100ul of mixed solution for each, and performing a P ℃ R program: 5 minutes at 95 ℃ (30 seconds at 95 ℃, 30 seconds at 55 ℃, 2 minutes at 72 ℃) X25 cycles, 5 minutes at 72 ℃, and an infinite at 4 ℃

5) Transferring the emulsion PCR product to a 1.5ml tube, adding 166ul of water saturated ether, carrying out vortex oscillation for 30 seconds, centrifuging at 12000rpm for 10 minutes, transferring a lower-layer liquid phase, standing at room temperature for 10 minutes until the ether is volatilized, extracting and purifying the liquid-phase product by adopting a phenol-chloroform method, and then precipitating with ethanol overnight to recover the product.

6) High fidelity PCR secondary amplification product

Performing PCR secondary amplification by using the product obtained in the step 4) as a template

The PCR procedure was as follows: 5 minutes at 95 ℃ and 5 minutes at 4 ℃ with 20 cycles of X (30 seconds at 95 ℃, 30 seconds at 62 ℃ and 2 minutes at 72 ℃) for 5 minutes at 72 DEG C

Taq _ F primer: ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACG (SEQ ID NO. 11)

Taq _ R primer: TTCCTTCGCAGATAACCAGTCTTCCCCTATGCCAACTTCGAC (SEQ ID NO. 12)

7) The PCR product was purified using a DNA product purification recovery kit and then religated with the pET28a expression vector. Thus completing one round of screening

8) The transformants religated with the pET28a vector were subjected to repetition of the steps (1) to (6), the conditions of emulsion PCR were changed in accordance with the procedure of the following table, and the selection pressure was gradually added to the mutant pool

And (3) second screening: 5 minutes at 95 ℃ (30 seconds at 95 ℃, 30 seconds at 55 ℃, 1.5 minutes at 72 ℃) X25 cycles, 5 minutes at 72 ℃, and an infinite at 4 ℃

And (3) third screening: 5 minutes at 95 ℃ (30 seconds at 95 ℃, 30 seconds at 55 ℃,1 minute at 72 ℃) X20 cycles, 5 minutes at 72 ℃, and an infinite at 4 ℃

And fourth screening: 5 minutes at 95 ℃ (30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃) X15 cycles, 5 minutes at 72 ℃, and an infinite at 4 ℃

After 4 rounds of selection, the resulting Taq enzyme mutant transformants were subjected to the high throughput screening of example 3

Example 3: high throughput screening of Taq enzyme mutants

384 single clones were randomly selected from the mutation library obtained in example 2, and after culturing and inducible expression, the amplification activity was tested by high throughput PCR, and 20 mutants having high amplification activity were selected from them. The method comprises the following specific steps:

1) selecting 384 monoclonals, inoculating into LB culture medium, culturing at 37 deg.C for 6 hr, adding isopropyl thiogalactoside (IPTG) with final concentration of 0.1mM, and inducing culturing at 37 deg.C for 3 hr

2) After the induction culture, the cells were collected by centrifugation, and a lysate (50Mm Tris, 50Mm NaCl, 5% glycerol pH8.5) containing 0.1mg/ml of lysozyme was added thereto to resuspend the cells, incubate them at 37 ℃ for 10 minutes, and heat them at 75 ℃ for 30 minutes. Then, the mixture was centrifuged at 12000rpm for 10 minutes to obtain a supernatant.

3) Taking a 96-well PCR plate, and adding the following reaction components into each well

10XTaq enzyme reaction solution (100mM Tris, 500mM KCl, 100mM (NH4)2SO4, pH8.0)2ul

PCR procedure: 5 minutes at 95 ℃ and 20 cycles of X (30 seconds at 95 ℃, 30 seconds at 62 ℃ and 60 seconds at 72 ℃) and 4C ∞

5ul of PCR products were subjected to agarose gel electrophoresis, and the yields of PCR products of supernatants prepared from the respective monoclones were compared to select 20 monoclones having the highest yield. The amplification yield of each mutant was 1.2-fold and 2-fold higher than that of the wild type.

Example 4: identification of dominant Taq enzyme mutant mutation site

DNA sequence sequencing of the Taq enzyme mutant selected in example 3 was performed to determine the mutation status of the amino acid sequence thereof, and the high frequency mutation site and the mutation form thereof were counted.

TABLE 1

Mutant numbering	Mutant amino acids
		1	E507A、K508L、E734E、F749K
2	K508L、V453A、R737K
		3	E734G
4	F749G、K508L、L764K
		5	E507Q、T757S
6	H785G
		7	S624T、F749V
8	E734F、F749V
		9	K508L、R737W、Y672R
10	E507H、H785L
		11	A518Q、E734M
12	F495R、F749T
		13	K508L、F749T、E734F
14	R737P、S624K
		15	T757W、V453G、E507M
16	F749E、H785G、F495G
		17	E734F、Y672P
18	T509L、H785K
		19	E734G、T757S、L764Q
20	K508L、V453A、A518Q

Sequencing 20 mutants with better amplification activity, and counting the amino acid mutation conditions as shown in the table above, it can be seen that: v453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764 and H785 occur in high-frequency repetition of 20 mutants, and the mutation of the mutant has a remarkable effect on the amplification activity of Taq enzyme.

Example 5 Mixed assay of Taq enzyme mutant with wild type Taq enzyme

Mixing the prepared Taq enzyme mutant and wild Taq enzyme according to a set proportion respectively, and then carrying out performance test.

Some representative mixed enzymes and test results are listed below.

TABLE 2

The concentration of each mixed enzyme is 10U/ul. According to the activity of the antibody: taq enzyme activity 1: 1 (supplied by Shenzhen Fenpengcheng Biotechnology Co., Ltd.), incubated at 37 ℃ for 30 minutes and stored at-20 ℃.

Using 2019 novel coronavirus (2019-nCoV) ORF1ab N nucleic acid detection kit (PCR-fluorescent probe method) (Daan Gene GmbH, Zhongshan university, national institutes of record 20203400749), reaction solution B was prepared using the above 27 mixed enzymes in the following manner to replace reaction solution B in the kit, and wild type and corresponding Taq mutant enzymes were used as negative control groups.

Reaction conditions are as follows: 2 min at 55 ℃, 2 min at 95 ℃ (15 sec at 95 ℃, fluorescence reading at 15 sec at 65 ℃) x 40 cycles, the Ct value of wild-type Taq enzyme amplified ORF1ab is 37.23, and the other Ct values are as follows:

TABLE 3

From the above results, it can be seen that the amplification performance of the positive reference of the novel coronavirus is significantly enhanced when Taq mutase 17 is mixed with wild type Taq enzyme compared with that of the wild type Taq and Taq mutant alone. Wherein, the Taq mutant 17 and the wild type Taq enzyme are prepared according to the following steps of 5:5 when they are mixed, the amplification effect is the best.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Daan Gen-Shaw Co Ltd of Zhongshan university

<120> DNA polymerase mixture for detection of novel coronavirus

<130> 020082

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60

catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120

gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180

gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240

tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300

gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360

gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420

gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480

tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540

gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600

gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660

ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720

ctgtcttggg acctggcgaa agttcgtact gatttgccgc tggaggtcga ctttgcgaag 780

cgtcgcgagc ccgatcgtga acgtcttcgc gcatttctgg agcgtttaga atttggctcc 840

ctgttgcatg agtttggttt gcttgaaagc ccgaaggcac ttgaggaagc tccttggcct 900

ccgcctgagg gcgcttttgt cggatttgtc ttgagccgta aagaaccgat gtgggcggac 960

ttactggccc ttgctgctgc tcgtgggggt cgcgtgcatc gcgcaccgga gccatacaaa 1020

gcacttcgtg accttaaaga agcccgtggc ttgttggcaa aagatttaag tgtcctggct 1080

ttacgcgagg gcttgggctt accaccggga gatgatccga tgcttttggc ctatctgctg 1140

gacccgagca acacgactcc agagggcgtt gcccgtcgtt atggcggaga atggacggag 1200

gaggcgggag agcgcgcagc gttaagcgag cgtctgtttg ctaatctgtg gggacgctta 1260

gagggagagg agcgcctgtt gtggttgtac cgtgaagtgg aacggccgct gagtgcagtg 1320

ttagctcaca tggaagcaac cggggtgcgg ctggacgttg cgtatttgcg tgcgctgtcg 1380

ttagaggtcg cggaggaaat agcccgtctg gaggccgaag tattccgttt ggctggccat 1440

cctttcaacc tgaacagtcg ggatcagctg gaacgtgtac tttttgatga actggggctg 1500

cccgccatcg gcaaaaccga aaaaaccggc aaacgtagca cctctgcggc agtgctggaa 1560

gcgttacgtg aagctcatcc gattgtggag aaaattctgc aatatcgcga attgacgaaa 1620

ctgaagagca cctatattga tccgctgcca gacttaattc acccccgtac cggacggttg 1680

catacccgct tcaaccagac cgcgacggcg acagggcggc tgagtagcag cgatccgaac 1740

ctgcaaaaca ttcccgtgcg taccccgctg ggtcagcgta ttcgccgtgc tttcattgcc 1800

gaggaaggct ggctgctggt cgcgctggac tactcgcaaa tcgaattgcg tgtgttggcc 1860

cacctgtcgg gcgacgaaaa cttaatacgc gtgtttcaag aaggtcgtga catacatact 1920

gaaaccgcgt cctggatgtt tggagtccca cgggaggctg tcgatcctct tatgcgtcgt 1980

gccgccaaaa caattaactt cggagttctg tacggcatgt cggcacatcg tttatcacag 2040

gaactggcga ttccgtatga agaagcgcag gccttcatag aacgttattt ccaatcattc 2100

cccaaggtgc gggcctggat tgagaagacc ctggaagagg gccgtcgtcg tggctatgta 2160

gagactctgt tcggacgtcg gcggtatgta cccgatcttg aggcccgtgt gaagtccgtt 2220

cgtgaggcag cagaacgtat ggcgtttaac atgccagtcc agggcacagc ggcggacctg 2280

atgaaattag ctatggttaa gctgtttccg cgtttggaag aaatgggcgc tcgtatgctg 2340

ttacaggttc atgacgagtt agtattagaa gcaccgaagg agcgtgccga agccgtggcc 2400

cggttagcca aagaggtaat ggaaggcgtc tacccccttg cagtcccgct tgaagtcgaa 2460

gttggcatag gggaagactg gttatctgcg aaggaa 2496

<210> 2

<211> 832

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu

1 5 10 15

Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly

20 25 30

Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala

35 40 45

Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val

50 55 60

Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly

65 70 75 80

Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu

85 90 95

Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu

100 105 110

Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys

115 120 125

Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp

130 135 140

Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly

145 150 155 160

Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro

165 170 175

Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn

180 185 190

Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu

195 200 205

Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu

210 215 220

Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys

225 230 235 240

Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val

245 250 255

Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe

260 265 270

Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu

275 280 285

Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly

290 295 300

Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp

305 310 315 320

Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro

325 330 335

Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu

340 345 350

Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro

355 360 365

Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn

370 375 380

Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu

385 390 395 400

Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu

405 410 415

Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu

420 425 430

Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly

435 440 445

Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala

450 455 460

Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His

465 470 475 480

Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp

485 490 495

Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg

500 505 510

Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile

515 520 525

Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr

530 535 540

Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu

545 550 555 560

His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser

565 570 575

Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln

580 585 590

Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala

595 600 605

Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly

610 615 620

Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr

625 630 635 640

Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro

645 650 655

Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly

660 665 670

Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu

675 680 685

Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg

690 695 700

Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val

705 710 715 720

Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg

725 730 735

Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro

740 745 750

Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu

755 760 765

Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His

770 775 780

Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala

785 790 795 800

Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro

805 810 815

Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu

820 825 830

<210> 3

<211> 1269

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60

catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120

gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180

gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240

tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300

gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360

gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420

gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480

tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540

gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600

gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660

ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720

ctgtcttggg acctggcgaa agttcgtact gatttgccgc tggaggtcga ctttgcgaag 780

cgtcgcgagc ccgatcgtga acgtcttcgc gcatttctgg agcgtttaga atttggctcc 840

ctgttgcatg agtttggttt gcttgaaagc ccgaaggcac ttgaggaagc tccttggcct 900

ccgcctgagg gcgcttttgt cggatttgtc ttgagccgta aagaaccgat gtgggcggac 960

ttactggccc ttgctgctgc tcgtgggggt cgcgtgcatc gcgcaccgga gccatacaaa 1020

gcacttcgtg accttaaaga agcccgtggc ttgttggcaa aagatttaag tgtcctggct 1080

ttacgcgagg gcttgggctt accaccggga gatgatccga tgcttttggc ctatctgctg 1140

gacccgagca acacgactcc agagggcgtt gcccgtcgtt atggcggaga atggacggag 1200

gaggcgggag agcgcgcagc gttaagcgag cgtctgtttg ctaatctgtg gggacgctta 1260

gagggagag 1269

<210> 4

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

atatcatatg cgtggcatgc tgccgctttt 30

<210> 5

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gcatgaattc cgtctcctct ccctctaagc 30

<210> 6

<211> 1236

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ggagaggagc gcctgttgtg gttgtaccgt gaagtggaac ggccgctgag tgcagtgtta 60

gctcacatgg aagcaaccgg ggtgcggctg gacgttgcgt atttgcgtgc gctgtcgtta 120

gaggtcgcgg aggaaatagc ccgtctggag gccgaagtat tccgtttggc tggccatcct 180

ttcaacctga acagtcggga tcagctggaa cgtgtacttt ttgatgaact ggggctgccc 240

gccatcggca aaaccgaaaa aaccggcaaa cgtagcacct ctgcggcagt gctggaagcg 300

ttacgtgaag ctcatccgat tgtggagaaa attctgcaat atcgcgaatt gacgaaactg 360

aagagcacct atattgatcc gctgccagac ttaattcacc cccgtaccgg acggttgcat 420

acccgcttca accagaccgc gacggcgaca gggcggctga gtagcagcga tccgaacctg 480

caaaacattc ccgtgcgtac cccgctgggt cagcgtattc gccgtgcttt cattgccgag 540

gaaggctggc tgctggtcgc gctggactac tcgcaaatcg aattgcgtgt gttggcccac 600

ctgtcgggcg acgaaaactt aatacgcgtg tttcaagaag gtcgtgacat acatactgaa 660

accgcgtcct ggatgtttgg agtcccacgg gaggctgtcg atcctcttat gcgtcgtgcc 720

gccaaaacaa ttaacttcgg agttctgtac ggcatgtcgg cacatcgttt atcacaggaa 780

ctggcgattc cgtatgaaga agcgcaggcc ttcatagaac gttatttcca atcattcccc 840

aaggtgcggg cctggattga gaagaccctg gaagagggcc gtcgtcgtgg ctatgtagag 900

actctgttcg gacgtcggcg gtatgtaccc gatcttgagg cccgtgtgaa gtccgttcgt 960

gaggcagcag aacgtatggc gtttaacatg ccagtccagg gcacagcggc ggacctgatg 1020

aaattagcta tggttaagct gtttccgcgt ttggaagaaa tgggcgctcg tatgctgtta 1080

caggttcatg acgagttagt attagaagca ccgaaggagc gtgccgaagc cgtggcccgg 1140

ttagccaaag aggtaatgga aggcgtctac ccccttgcag tcccgcttga agtcgaagtt 1200

ggcatagggg aagactggtt atctgcgaag gaataa 1236

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ggagaggagc gcctgttgtg gttgt 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ttattccttc gcagataacc agtct 25

Claims (10)

1. A DNA polymerase mixture comprising a wild-type DNA polymerase and a mutant DNA polymerase; wherein the amino acid sequence of the wild type DNA polymerase is shown as SEQ ID NO. 2;

the mutant DNA polymerase is mutant 17: the mutation is performed on the basis of the wild-type DNA polymerase shown in SEQ ID No. 2 and the mutant 17 has the following mutation sites: E734F, Y672P.

2. The DNA polymerase mixture of claim 1, wherein the mutation sites of mutant 17 are: E734F, Y672P.

3. The DNA polymerase mixture of claim 1, wherein the ratio of the enzyme activities of the wild-type DNA polymerase and the mutant DNA polymerase in the DNA polymerase mixture is 1-9: 9: 1.

4. the DNA polymerase mixture of claim 4 wherein the ratio of the enzyme activities of the wild-type DNA polymerase and the mutant DNA polymerase in the DNA polymerase mixture is 2-8: 8: 2.

5. the DNA polymerase mixture of claim 5, wherein the ratio of the enzyme activities of the wild-type DNA polymerase and the mutant DNA polymerase in the DNA polymerase mixture is 3-7: 7: 3.

6. the DNA polymerase mixture of claim 5 wherein the ratio of enzymatic activities of the wild-type DNA polymerase and the mutant DNA polymerase in the DNA polymerase mixture is 5: 5.

7. a kit comprising the DNA polymerase mixture of claim 1.

8. The kit of claim 7, wherein the kit further comprises a PCR buffer, a reverse transcriptase, and/or dNTPs.

9. A method of PCR amplification, the method comprising the steps of:

(1) providing a nucleic acid sample of an object to be detected;

(2) preparing a PCR reaction system and carrying out PCR reaction:

wherein in step (2), the DNA polymerase mixture of the first aspect of the invention is used to catalyze the polymerization of substrate dNTP molecules to form progeny DNA.

10. The method of claim 9, wherein the nucleic acid sample comprises cDNA of the novel coronavirus SARS-CoV-2.