Mixture for improving amplification platform and application thereof

Abstract

The invention provides a mixture for improving an amplification platform and application thereof, belongs to the technical field of biology, and particularly relates to a mixture containing a single-stranded DNA binding protein multimer, and the mixture can improve the amplification platform of an amplification reaction and can stabilize the amplification performance of an amplification reagent in a pressurized state.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mixture containing a single-stranded DNA binding protein polymer, wherein the mixture can improve an amplification platform of a real-time fluorescence quantitative PCR reaction, stabilize the amplification performance of a full premix reagent under a pressurized state, and improve the storage stability of the amplification reagent. Background Art

[0002] One-step reverse transcription polymerase chain reaction (RT-qPCR) is a molecular biology technique used to quickly, accurately and sensitively detect and quantify target RNA. It combines two key steps, reverse transcription and real-time fluorescence quantitative PCR, and can reverse transcribe RNA into cDNA in the same reaction, and quantify the initial amount of the target gene by real-time monitoring of the fluorescence signal during the PCR process, greatly simplifying the experimental operation process and reducing the time and cost of reagents and operations.

[0003] One-step RT-qPCR has a wide range of applications in many fields, such as studying gene regulation, epigenetics, and biological evolution. Although one-step RT-qPCR reagents perform well in rapid, accurate, and sensitive detection of target RNA, there are still some potential shortcomings. First, the primer dimer problem remains a challenge, especially after the reagents have been stored for a long time or pressurized at high temperature. The formation of primer dimers leads to loss of reagent performance, affecting amplification efficiency and accuracy. Secondly, for complex samples, such as mixtures containing multiple RNA or DNA molecules, nonspecific amplification may occur, thereby affecting the accuracy of quantitative results. In addition, one-step RT-qPCR has high requirements for reaction conditions and primer design, and requires repeated optimization and verification to ensure the repeatability and reliability of the experiment. In the face of these challenges, future improvements may include optimization of primer design, improvement of reagent storage conditions, and more intelligent experimental systems to further improve the performance and application range of one-step RT-qPCR reagents.

[0004] At present, the main direction of optimizing the performance of PCR amplification reagents is to reduce the generation of non-specific products in the reaction, such as introducing heat-labile blocking groups by chemical methods to block the polymerization activity of Taq enzyme at room temperature, blocking Taq enzyme activity by screening specific antibodies and ligands, or modifying the reaction primers to achieve the purpose of reducing non-specific amplification. However, these methods affect the balance of the reaction while reducing non-specific products, increase the cost of using RT-qPCR detection reagents, especially for primers. Different modifications or transformations greatly increase the complexity of primer synthesis, resulting in some methods cannot be applied on a large scale.

[0005] Another low-cost method is to add different additives to the reaction reagents to improve the performance of the reagents, such as Mg 2+ Reversibly bound polyanionic additives, polypeptide fragments, or modified random oligonucleotides, glutamic acid and its salts, and various organic additives such as DMSO, betaine and formamide. Another type of additive is to add different functional proteins to the reaction system to improve the reaction performance: such as adding DNA helicase to prevent random annealing of primers, and adding proteins that can detect and eliminate nucleic acid molecules containing errors, such as DNA polymerase, mismatch endonuclease, mismatch binding protein, etc. Summary of the invention

[0006] The present application is beneficial to improving the amplification platform of real-time fluorescence quantitative PCR by adding single-stranded DNA binding protein polymers to the amplification system, and can stabilize the amplification performance of the full premix reagent under pressurized state and improve the storage stability of the amplification reagent.

[0007] In a first aspect, the present application provides a mixture comprising a DNA polymerase, a buffer substance, a metal salt and a single-stranded DNA binding protein polymer.

[0008] In some embodiments, the single-stranded DNA binding protein polymer comprises at least two single-stranded DNA binding protein monomers. In some embodiments, the single-stranded DNA binding protein polymer is a dimer, trimer or tetramer. In some embodiments, the single-stranded DNA binding protein monomers in the single-stranded DNA binding protein polymer are of the same species. In some embodiments, the single-stranded DNA binding protein monomers in the single-stranded DNA binding protein polymer are of different species. In some embodiments, the single-stranded DNA binding protein monomer comprises Escherichia coli single-stranded binding protein, Thermus thermophilus single-stranded binding protein, Methanococcus jannaschii single-stranded binding protein, T4GP32 protein, Cspa cold-sensitive protein, Muts protein and / or RecA protein; preferably, the single-stranded DNA binding protein monomer comprises Escherichia coli single-stranded binding protein, T4GP32 protein and / or Cspa cold-sensitive protein.

[0009] In some embodiments, the single-stranded DNA binding protein polymer comprises an Escherichia coli single-stranded binding protein, a T4GP32 protein and / or a Cspa cold-sensitive protein. In some embodiments, the single-stranded DNA binding protein polymer is a Cspa cold-sensitive protein polymer. In some embodiments, the single-stranded DNA binding protein polymer is an Escherichia coli single-stranded binding protein polymer. In some embodiments, the single-stranded DNA binding protein polymer is a T4GP32 protein polymer. In some embodiments, the single-stranded DNA binding protein polymer is a Cspa cold-sensitive protein and a T4GP32 protein polymer. In some embodiments, the single-stranded DNA binding protein polymer is a T4GP32 protein and an Escherichia coli single-stranded binding protein polymer. In some embodiments, the single-stranded DNA binding protein polymer is a Cspa cold-sensitive protein, a T4GP32 protein and an Escherichia coli single-stranded binding protein polymer.

[0010] In some embodiments, the single-stranded DNA binding protein multimer comprises any one or more of the proteins shown in SEQ ID NOs: 37-50. In some embodiments, the single-stranded DNA binding protein dimer comprises any one or more of the proteins shown in SEQ ID NOs: 37-40. In some embodiments, the single-stranded DNA binding protein trimer comprises any one or more of the proteins shown in SEQ ID NOs: 41-47. In some embodiments, the single-stranded DNA binding protein tetramer comprises any one or more of the proteins shown in SEQ ID NOs: 48-50.

[0011] In some embodiments, the molar ratio of the single-stranded DNA binding protein polymer to the single-stranded DNA in a single reaction is 0.5-20; preferably 1-10; and more preferably 2-5.

[0012] In some embodiments, the single-stranded DNA binding protein multimer is obtained by direct expression in bacteria; preferably, the single-stranded DNA binding protein multimer is obtained by expression in Escherichia coli. In some embodiments, the single-stranded DNA binding protein multimer is obtained by in vitro cross-linking polymerization of single-stranded DNA binding protein monomers. In some embodiments, there is a connecting part between the monomers in the single-stranded DNA binding protein multimer, and the connecting part consists of 1-25 arbitrary amino acid sequences; preferably, the connecting part consists of 5-20 arbitrary amino acid sequences; preferably, the connecting part consists of 8-15 arbitrary amino acid sequences.

[0013] In some embodiments, the DNA polymerase comprises a DNA-dependent DNA polymerase and / or an RNA-dependent DNA polymerase. In some embodiments, the DNA-dependent DNA polymerase comprises one or more of Taq DNA polymerase, Pfu DNA polymerase, and Bst DNA polymerase. In some embodiments, the RNA-dependent DNA polymerase comprises one or more of MMLV reverse transcriptase, AMV reverse transcriptase, and HIV reverse transcriptase. In some embodiments, the mixture comprises Taq DNA polymerase, a buffer substance, a metal salt, and a single-stranded DNA binding protein polymer, wherein the single-stranded DNA binding protein polymer comprises one or more of SEQ ID NOs: 37-50; optionally, the mixture also comprises MMLV reverse transcriptase.

[0014] In some embodiments, the buffer substance comprises at least one of Tris, Hepes, Pipes, Taps or Tricine; preferably, the buffer substance is Tris. In some embodiments, the metal salt comprises one or more of magnesium salt, potassium salt, manganese salt, sodium salt, calcium salt or ammonium salt; preferably, the metal salt comprises magnesium salt and / or potassium salt. In some embodiments, the mixture comprises Taq DNA polymerase, Tris, MgCl2, KCl, single-stranded DNA binding protein polymer, wherein the single-stranded DNA binding protein polymer comprises one or more of SEQ ID NO: 37-50; optionally, the mixture also comprises MMLV reverse transcriptase.

[0015] In some embodiments, the mixture further comprises deoxyribonucleoside triphosphates, and the deoxyribonucleoside triphosphates comprise one or more of dATP, dCTP, dTTP, dGTP, and dUTP. In some embodiments, the deoxyribonucleoside triphosphates are a mixture of dATP, dCTP, dTTP, and dGTP; preferably, the deoxyribonucleoside triphosphates are a mixture of dATP, dCTP, dTTP, dGTP, and dUTP. In some embodiments, the mixture comprises Taq DNA polymerase, Tris, MgCl2, KCl, dATP, dTTP, dCTP, dGTP, and a single-stranded DNA binding protein polymer, wherein the single-stranded DNA binding protein polymer comprises one or more of SEQ ID NOs: 37-50; optionally, the mixture further comprises MMLV reverse transcriptase.

[0016] In some embodiments, the mixture further comprises a primer, a probe or a fluorescent dye. In some embodiments, the probe is a TaqMan probe. In some embodiments, the fluorescent dye comprises one of SYBR GreenⅠ, LC GreenPLUS or EvaGreen; preferably, the fluorescent dye is SYBR GreenⅠ. In some embodiments, the mixture comprises Taq DNA polymerase, Tris, MgCl2, KCl, dATP, dTTP, dCTP, dGTP, primer, TaqMan probe or SYBR GreenⅠ, single-stranded DNA binding protein polymer, wherein the single-stranded DNA binding protein polymer comprises one or more of SEQ ID NO: 37-50; optionally, the mixture further comprises MMLV reverse transcriptase.

[0017] In some embodiments, the mixture further comprises uracil-DNA glycosylase; preferably, the mixture comprises dATP, dCTP, dTTP, dGTP, dUTP, Taq DNA polymerase, uracil-DNA glycosylase, Tris, MgCl2, KCl, primers, TaqMan probe or SYBR GreenⅠ, and a single-stranded DNA binding protein polymer, wherein the single-stranded DNA binding protein polymer comprises one or more of those shown in SEQ ID NOs: 37-50; optionally, the mixture further comprises MMLV reverse transcriptase.

[0018] In some embodiments, the mixture further comprises a template, which is DNA and / or RNA; preferably, the mixture comprises DNA and / or RNA, dATP, dCTP, dTTP, dGTP, dUTP, Taq DNA polymerase, uracil-DNA glycosylase, Tris, MgCl2, KCl, primers, TaqMan probes or SYBR GreenⅠ, and a single-stranded DNA binding protein polymer, wherein the single-stranded DNA binding protein polymer comprises one or more of those shown in SEQ ID NOs: 37-50; optionally, the mixture further comprises MMLV reverse transcriptase.

[0019] The second aspect of the present application provides a single-stranded DNA binding protein polymer, which comprises Escherichia coli single-stranded DNA binding protein, T4GP32 protein and / or Cspa cold-sensitive protein.

[0020] In some embodiments, the monomers in the single-stranded DNA binding protein multimer are of the same species. In some embodiments, the single-stranded DNA binding protein multimer is a Cspa cold-sensitive protein multimer. In some embodiments, the single-stranded DNA binding protein multimer is an Escherichia coli single-stranded binding protein multimer. In some embodiments, the single-stranded DNA binding protein multimer is a T4GP32 protein multimer. In some embodiments, the single-stranded DNA binding protein multimer comprises any one or more of the proteins shown in SEQ ID NOs: 37-39, SEQ ID NOs: 41-43, and SEQ ID NOs: 48-50.

[0021] In some embodiments, the monomers in the single-stranded DNA binding protein multimer are of different species. In some embodiments, the single-stranded DNA binding protein multimer is a Cspa cold-sensitive protein and a T4GP32 protein multimer. In some embodiments, the single-stranded DNA binding protein multimer is a T4GP32 protein and an Escherichia coli single-stranded binding protein multimer. In some embodiments, the single-stranded DNA binding protein multimer is a Cspa cold-sensitive protein, a T4GP32 protein and an Escherichia coli single-stranded binding protein multimer. In some embodiments, the single-stranded DNA binding protein multimer comprises any one or more of the proteins shown in SEQ ID NO: 40, SEQ ID NO: 44-46.

[0022] The third aspect of the present application provides a polymerase chain amplification reaction method, which comprises the step of adding the single-stranded DNA binding protein polymer described in the second aspect of the present application to a reaction system.

[0023] In some embodiments, the molar ratio of the single-stranded DNA binding protein polymer to the single-stranded DNA in a single reaction is 0.5-20; preferably 1-10; and more preferably 2-5.

[0024] The fourth aspect of the present application provides a kit, which comprises components that can constitute the mixture described in the first aspect of the present application.

[0025] The fifth aspect of the present application provides another kit, which comprises the single-stranded DNA binding protein polymer described in the second aspect of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A :The results of Cspa monomer and polymer in RT-qPCR reaction;

[0027] Figure 1B :The results of T4GP32 monomer and polymer in RT-qPCR reaction;

[0028] Figure 1C :The results of Ecoli-SSB monomer and polymer in RT-qPCR reaction;

[0029] Figure 2A :RT-qPCR detection results of heterologous single-stranded DNA binding protein polymers in H-07 system;

[0030] Figure 2B :RT-qPCR detection results of heterologous single-stranded DNA binding protein polymers in M-52 system;

[0031] Figure 3A :The results of Cspa dimer and its monomer in the pressure reaction of one-step RT-qPCR full premix reagent;

[0032] Figure 3B :The results of Cspa-T4GP32 dimer and its monomer in the one-step RT-qPCR full premix reagent pressurization reaction;

[0033] Figure 3C :The results of T4GP32-Ecoli-SSB-T4GP32 trimer and its monomer in the one-step RT-qPCR premix reagent pressurization reaction.

[0034] Figure 4A :qPCR detection results of different single-stranded DNA binding protein monomers and polymers in H-02 system;

[0035] Figure 4B :qPCR detection results of different single-stranded DNA binding protein monomers and polymers in H-17 system;

[0036] Figure 4C :qPCR detection results of different single-stranded DNA binding protein monomers and polymers in M-51 system;

[0037] Figure 5A : Experimental results of Cspa dimer and monomer in the pressurized reaction of qPCR full premix reagent;

[0038] Figure 5B : Experimental results of Cspa-T4GP32 dimer and monomer in the pressurized reaction of qPCR full premix reagent;

[0039] Fig. 6A : RT-qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 1;

[0040] Figure 6B : RT-qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 4;

[0041] Figure 6C: RT-qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 5;

[0042] Fig. 7A : qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 1;

[0043] Figure 7B : qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 2;

[0044] Figure 7C : qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 3. DETAILED DESCRIPTION

[0045] The technical solution of the present application is further explained below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are merely simple examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0046] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0047] Example 1: Material preparation

[0048] 1. Sample preparation

[0049] In this example, Nanjing Novozyme Biotechnology Co., Ltd. Cell / Tissue TotalRNA Isolation Kit (Vazyme, RC101) was used to extract RNA from Hela cells and hybridoma cells. The concentrations of the extracted RNA were 2459 ng / μl and 1890 ng / μl, respectively. After detection by qPCR reagent without reverse transcriptase, there was no DNA contamination and subsequent tests were possible. The FastPure Blood / Cell / Tissue / Bacteria DNA Isolation Mini Kit (Vazyme, DC112) DNA extraction kit from Nanjing Novezan Biotech Co., Ltd. was used to extract genomic DNA from Hela cells and hybridoma cells. The concentrations of the extracted DNA were 3687 ng / μl and 2658 ng / μl, respectively.

[0050] 2. Primer design

[0051] qPCR primers and probes were designed based on the human DCT (XM_017020401.3), MYC (NM_002467) and NW_021160015 gene sequences, and mouse Ncor2 (XM_030254302.1), Zbtb20 (NM_001393397.1), Chd7 (XM_006538005.5) and NC_000067.7 gene sequences. The specific sequences are shown in Table 1.

[0052] Table 1: qPCR primer and probe sequences for human and mouse targets in Example 1

[0053]

[0054]

[0055] Among them, the H series sequence name is human gene target, and the M series sequence name is mouse gene target. In Example 2-4, when the test system is H-09 or H-07, the added template is Hela cell RNA, and when the test system is M-5, M-51, M-52 or M-54, the added template is hybridoma cell RNA. In Example 5-6, when the sequencing system is H-02 or H-17, the added template is Hela cell DNA, and when the test system is M-13, M-15 or M-18, the added template is hybridoma cell DNA.

[0056] 3. Preparation of premixed reaction solutions A, C, E, F (excluding primers and probes) and premixed reaction solutions B and D (including primers and probes)

[0057] Table 2 is the preparation scheme of the complete premixed reaction solution A. Various one-step RT-qPCR complete premixed reaction solutions A were prepared according to Table 2, wherein the component concentrations of the "5×RNA amplification system" were: 250mmol / L Tris, 250mmol / L KAC, 100mmol / L KCl, 1.5mmol / LdNTPs, and 20mmol / LMgCl2.

[0058] Table 2: Full premixed reaction solution A

[0059]

[0060]

[0061] Table 3 is the preparation scheme of the complete premixed reaction solution C. Various qPCR complete premixed reaction solutions C were prepared according to Table 3, wherein the component concentrations of the "5×DNA amplification system" were: 200mmol / L Tris, 100mmol / L NH4AC, 300mmol / L KCl, 2mmol / L dNTPs, and 20mmol / LMgCl2.

[0062] Table 3: Full premixed reaction solution C

[0063]

[0064] Table 4 shows five different ion buffer component preparation schemes used to verify the effects of single-stranded DNA binding proteins on qPCR and RT-qPCR under different buffer conditions.

[0065] Table 4: Preparation scheme of different ion buffer components

[0066]

[0067]

[0068] Wherein, TEAC is tetraethylammonium chloride, and TMAC is tetramethylammonium chloride. Prepare the fully premixed reaction solution E according to Table 2, wherein the "5× RNA amplification system" is buffer 1, buffer 4, and buffer 5 in Table 4; prepare the fully premixed reaction solution F according to Table 3, wherein the "5× DNA amplification system" is buffer 1, buffer 2, and buffer 3 in Table 4.

[0069] Note: The fully premixed reaction solutions A, B, and E contain reverse transcriptase and are suitable for one-step RT-qPCR systems. The fully premixed reaction solutions C, D, and F only contain DNA polymerase and are suitable for DNAqPCR systems.

[0070] The single-stranded DNA binding proteins in the examples of the present application include three types: cold-sensitive protein Cspa, T4GP32 and single-stranded DNA binding protein derived from Escherichia coli (Ecoli-SSB). 16 fully premixed reaction solutions A, 6 fully premixed reaction solutions B, 7 fully premixed reaction solutions C, 4 fully premixed reaction solutions D, 15 fully premixed reaction solutions E and 15 fully premixed reaction solutions F were prepared according to the following description.

[0071] ① According to Table 2, a fully premixed reaction solution A without adding any single-stranded DNA binding protein was prepared, named fully premixed reaction solution 1, as the experimental control group.

[0072] ② Prepare a fully premixed reaction solution A to which a cold-sensitive protein Cspa monomer or a polymer thereof is added according to Table 2 and name them as fully premixed reaction solutions 2-5 respectively: prepare fully premixed reaction solution 2 by adding 6 μg of cold-sensitive protein Cspa monomer (SEQ ID NO: 34) to each reaction solution; prepare fully premixed reaction solution 3 by adding 1 μg of cold-sensitive protein Cspa dimer (SEQ ID NO: 37) to each reaction solution; prepare fully premixed reaction solution 4 by adding 1 μg of cold-sensitive protein Cspa trimer (SEQ ID NO: 41) to each reaction solution; prepare fully premixed reaction solution 5 by adding 1 μg of cold-sensitive protein Cspa tetramer (SEQ ID NO: 48) to each reaction solution.

[0073] ③ Prepare a fully premixed reaction solution A to which T4GP32 single-stranded DNA binding protein monomer or multimer is added according to Table 2 and name them as fully premixed reaction solutions 6-9 respectively: prepare fully premixed reaction solution 6 by adding 6 μg T4GP32 monomer (SEQ ID NO: 35) to each reaction solution; prepare fully premixed reaction solution 7 by adding 1.5 μg T4GP32 dimer (SEQ ID NO: 38) to each reaction solution; prepare fully premixed reaction solution 8 by adding 1.5 μg T4GP32 trimer (SEQ ID NO: 42) to each reaction solution; prepare fully premixed reaction solution 9 by adding 1.5 μg T4GP32 tetramer (SEQ ID NO: 49) to each reaction solution.

[0074] ④ Prepare a fully premixed reaction solution A to which Ecoli-SSB single-stranded DNA binding protein monomer or polymer is added according to Table 2 and name them as fully premixed reaction solutions 10-13 respectively: prepare fully premixed reaction solution 10 by adding 6 μg Ecoli-SSB monomer (SEQ ID NO: 36) to each reaction solution; prepare fully premixed reaction solution 11 by adding 1 μg Ecoli-SSB dimer (SEQ ID NO: 39) to each reaction solution; prepare fully premixed reaction solution 12 by adding 1 μg Ecoli-SSB trimer (SEQ ID NO: 43) to each reaction solution; prepare fully premixed reaction solution 13 by adding 1 μg Ecoli-SSB tetramer (SEQ ID NO: 50) to each reaction solution.

[0075] ⑤ Prepare a fully premixed reaction solution A with the addition of different single-stranded DNA binding protein trimers according to Table 2 and name it as fully premixed reaction solutions 14-16: prepare fully premixed reaction solution 14 by adding 1 μg Cspa-T4GP32-Ecoli-SSB trimer (SEQ ID NO: 44) to each reaction solution; prepare fully premixed reaction solution 15 by adding 1 μg T4GP32-Cspa-Ecoli-SSB trimer (SEQ ID NO: 45) to each reaction solution; prepare fully premixed reaction solution 16 by adding 1 μg Ecoli-SSB-T4GP32-Cspa trimer (SEQ ID NO: 46) to each reaction solution.

[0076] ⑥ According to Table 2, 0.4 μl (10 μmol / L) upstream primer, 0.4 μl (10 μmol / L) downstream primer, 0.2 μl (10 μmol / L) corresponding probe and single-stranded DNA binding protein monomer or polymer were added to each reaction solution to prepare a fully premixed reaction solution B, wherein 6 μg Cspa protein monomer was added to prepare a fully premixed reaction solution 17, 1.5 μg Cspa protein dimer was added to prepare a fully premixed reaction solution 18, 1.5 μg Cspa-T4GP32 dimer (SEQ ID NO: 40) was added to prepare a fully premixed reaction solution 19, 6 μg T4GP32 protein monomer was added to prepare a fully premixed reaction solution 20, 1 μg T4GP32-Ecoli-SSB-T4GP32 trimer (SEQ ID NO: 47) was added to prepare a fully premixed reaction solution 21, and 6 μg Ecoli-SSB protein monomer was added to prepare a fully premixed reaction solution 22.

[0077] ⑦ According to Table 3, fully premixed reaction solutions C suitable for DNA qPCR system were prepared and named as fully premixed reaction solutions 23-29 respectively: 6 μg of cold-sensitive protein Cspa monomer (SEQ ID NO: 34) was added to each reaction solution to prepare fully premixed reaction solution 23; 6 μg of T4GP32 monomer (SEQ ID NO: 35) was added to each reaction solution to prepare fully premixed reaction solution 24; 6 μg of Ecoli-SSB monomer (SEQ ID NO: 36) was added to each reaction solution to prepare fully premixed reaction solution 25; 1 μg of cold-sensitive protein Cspa dimer (SEQ ID NO: 37) was added to each reaction solution to prepare fully premixed reaction solution 26; 1.5 μg of T4GP32 trimer (SEQ ID NO: 42) was added to each reaction solution to prepare fully premixed reaction solution 27; 1 μg of Ecoli-SSB tetramer (SEQ ID NO: 50) was added to each reaction solution to prepare fully premixed reaction solution 28; 1 ​​μg of Cspa-T4GP32-Ecoli-SSB trimer (SEQ ID NO: 44) was prepared into a fully premixed reaction solution 29.

[0078] ⑧ According to Table 3, 0.4 μl (10 μmol / L) upstream primer, 0.4 μl (10 μmol / L) downstream primer, 0.2 μl (10 μmol / L) corresponding probe and single-stranded DNA binding protein monomer or polymer were added to each reaction solution to prepare a fully premixed reaction solution D, wherein the fully premixed reaction solution 30 was added with 1.5 μg Cspa protein dimer, the fully premixed reaction solution 31 was added with 6 μg Cspa protein monomer, the fully premixed reaction solution 32 was added with 6 μg T4GP32 protein monomer, and the fully premixed reaction solution 33 was added with 1.5 μg Cspa-T4GP32 dimer (SEQ ID NO: 40).

[0079] ⑨ According to Tables 2 and 4, 15 fully premixed reaction solutions E were prepared and named as fully premixed reaction solutions 34-48: When buffer 1 in Table 4 was used as the "5× RNA amplification system" in Table 2, fully premixed reaction solutions 34-38 were prepared, wherein when no single-stranded DNA binding protein was added to the reaction system, it was fully premixed reaction solution 34; 6 μg of cold-sensitive protein Cspa monomer was added to each reaction solution to prepare fully premixed reaction solution 35; 6 μg of T4GP32 monomer was added to each reaction solution to prepare fully premixed reaction solution 36; 1 μg of cold-sensitive protein Cspa dimer was added to each reaction solution to prepare fully premixed reaction solution 37; 1.5 μg of A fully premixed reaction solution 38 of Cspa-T4GP32 dimer was prepared; when buffer 4 in Table 4 was used as the "5×RNA amplification system" in Table 2, a fully premixed reaction solution 39-43 was prepared without adding any single-stranded DNA binding protein to the reaction system, 6 μg of cold-sensitive protein Cspa monomer was added to each reaction solution, 6 μg of T4GP32 monomer was added to each reaction solution, 1 μg of cold-sensitive protein Cspa dimer was added to each reaction solution, and 1.5 μg of Cspa-T4GP32 dimer was added to each reaction solution; when buffer 5 in Table 4 was used as the "5×RNA amplification system" in Table 2, a fully premixed reaction solution 44-48 was prepared without adding any single-stranded DNA binding protein to the reaction system, 6 μg of cold-sensitive protein Cspa monomer was added to each reaction solution, 6 μg of T4GP32 monomer was added to each reaction solution, 1 μg of cold-sensitive protein Cspa dimer was added to each reaction solution, and 1.5 μg of Cspa-T4GP32 dimer was added to each reaction solution.

[0080] ⑩ According to Tables 3 and 4, 15 kinds of fully premixed reaction solutions F were prepared and named as fully premixed reaction solutions 49-63: When buffer 1 in Table 4 was used as the "5×DNA amplification system" in Table 2, no single-stranded DNA binding protein was added to the prepared reaction system, 6 μg of cold-sensitive protein Cspa monomer was added to each reaction solution, 6 μg of T4GP32 monomer was added to each reaction solution, 1 μg of cold-sensitive protein Cspa dimer was added to each reaction solution, and 1.5 μg of Cspa-T4GP32 dimer was added to each reaction solution; When buffer 2 in Table 4 was used as the "5×DNA amplification system" in Table 2, no single-stranded DNA binding protein was added to the prepared reaction system, 6 μg of cold-sensitive protein Cspa monomer was added to each reaction solution, 6 μg of T4GP32 monomer was added to each reaction solution, 1 μg of cold-sensitive protein Cspa dimer was added to each reaction solution, and 1.5 μg of fully premixed reaction solution of Cspa-T4GP32 dimer 54-58; when buffer 3 in Table 4 is used as the "5×DNA amplification system" in Table 2, the reaction system is prepared without adding any single-stranded DNA binding protein, 6 μg of cold-sensitive protein Cspa monomer is added to each reaction solution, 6 μg of T4GP32 monomer is added to each reaction solution, 1 μg of cold-sensitive protein Cspa dimer is added to each reaction solution, and 1.5 μg of Cspa-T4GP32 dimer is added to each reaction solution. Fully premixed reaction solution 59-63.

[0081] Among them, the single-stranded DNA binding protein monomers and polymers were prepared according to the following method: the corresponding nucleic acid sequence was designed according to the protein amino acid sequence, and all nucleic acid sequences were synthesized in Sangon Biotech (Shanghai) Co., Ltd. After synthesis, the nucleic acid sequence was connected to the pET28a expression vector and transformed into the Escherichia coli BL21 host for expression. After the monomers and polymers were overexpressed, centrifuged at 12000rpm for 10min, the supernatant was discarded, the cells were collected, the loading buffer was added to resuspend the cells, the cells were ultrasonically broken, the supernatant was collected by centrifugation, and the target protein was purified using an NTA chromatography column to obtain different single-stranded DNA binding protein monomers and polymers.

[0082] Example 2: Effect of the Same Single-stranded DNA Binding Protein Polymer on One-step RT-qPCR Reaction

[0083] According to the method in Example 1, 1-13 in the fully premixed reaction solution A was prepared, and 10 pg of the extracted total RNA and the corresponding primer probe were added to prepare single reaction systems 1-13. Each system was repeated twice, and the H-09 and M-51 genes were tested respectively. Reaction system 1-5 was the first group, reaction system 1 and reaction system 6-9 were the second group, and reaction system 1 and reaction system 10-13 were the third group. The effects of single-stranded DNA binding protein monomers and the same single-stranded DNA binding protein polymers on the one-step RT-qPCR reaction in the same reaction were detected.

[0084] The prepared reaction system was reacted according to Table 5.

[0085] Table 5: Reaction Procedure

[0086]

[0087] Experimental results analysis:

[0088] The experimental results are shown in Figures 1A-1C ,in Figure 1A The effect of Cspa monomer and polymer on RT-qPCR reaction. Figure 1B The effect of T4GP32 monomer and polymer on RT-qPCR reaction. Figure 1CThe effect of Ecoli-SSB monomer and its polymer on RT-qPCR reaction. As can be seen from Figure 1, after adding single-stranded DNA binding protein monomer or its polymer to the amplification reaction system, the amplification platform of RT-qPCR reaction is significantly improved compared with the control group without addition, among which Cspa polymer can increase the amplification platform by 80% to 300% compared with the control group without adding single-stranded DNA binding protein, especially in the M51 system, it can be increased by more than 200%, compared with the control group with added Cspa monomer, the amplification platform of the experimental group with added polymer can be increased by 40% to 100%, especially in the M51 system, it can be increased by more than 60%; T4GP32 monomer has an amplification platform of 20% to 50% compared with the control group without adding single-stranded DNA binding protein, and T4GP32 protein polymer can increase the amplification platform by more than 50% compared with the control group without adding single-stranded DNA binding protein, and can be increased by up to 235%, compared with the control group of T4GP32 monomer, Its amplification platform is improved by more than 30%, and can be increased by up to 123%; for Ecoli-SSB, after adding Ecoli-SSB polymers to the amplification reaction, the amplification platform of the reagent is improved by more than 150% compared with the control group without addition, and by more than 50% compared with the control group with Ecoli-SSB monomers. In addition, in the Ecoli-SSB protein experiment, with the increase in the number of monomers in the polymer, the improvement of the amplification platform of the reagent is more significant. For example, in the M51 system, compared with the system without adding single-stranded DNA binding protein, the amplification platform of the system with the addition of Ecoli-SSB dimer, trimer and tetramer is improved by 150%, 200% and 225%, respectively. Compared with the system with the addition of Ecoli-SSB monomers, the amplification platform of the system with the addition of Ecoli-SSB dimer, trimer and tetramer is improved by 66.7%, 100% and 119%, respectively.

[0089] The results showed that the amplification platform could be significantly improved by adding Cspa, T4GP32 or Ecoli-SSB polymers to the amplification reaction system, and in some systems, the more monomers there were in the polymer, the more the amplification platform was improved.

[0090] Example 3: Effect of heterologous single-stranded DNA binding protein polymers on one-step RT-qPCR reaction

[0091] According to the method in Example 1, 1, 2, 6, 10 and 14-16 in the fully premixed reaction solution A were prepared, and 10 pg of the extracted total RNA and the corresponding primer probes were added to prepare the corresponding reaction system. The experimental reaction was carried out according to Table 3. Two replicates were performed for each system to test the H-07 and M-52 genes respectively, and the effects of different single-stranded DNA binding protein monomers and heterologous polymers on the one-step RT-qPCR reaction in the same reaction were detected.

[0092] Experimental results analysis:

[0093] The experimental results are shown in Figure 2A and 2B ,in Figure 2A This is the result of the H-07 gene test. Figure 2B The results of M-52 gene testing. Figure 2A and Figure 2B It can be seen that compared with the control group without adding single-stranded DNA binding protein, after adding different single-stranded DNA binding protein monomers to the reaction system, the amplification platform of the reagent is improved by 40% to 70%. When the single-stranded DNA binding protein monomers from different sources form a multimer, the amplification platform can be improved by 110% to 200% compared with the reagent with only monomers added, and the connection order of each single-stranded DNA binding protein in the heteropolymer has little effect on the amplification platform improvement performance of the amplification reagent. In the H-07 gene detection, the improvement of the amplification platform by heterotrimers is between 110% and 130%, and the improvement of the amplification platform in the M-52 gene detection is more significant. Compared with the control group without adding single-stranded DNA binding protein, the improvement of the amplification platform is between 175% and 200%, and compared with the control group with different single-stranded DNA binding protein monomers, the amplification platform of heterotrimers is also improved by 60% to 120%. It can be seen that heterologous single-stranded DNA binding protein polymers can also effectively improve the amplification platform of amplification reagents, and are not limited to the type, number and order of single-stranded DNA binding protein monomers in the polymer.

[0094] Example 4: Effect of single-stranded DNA binding protein polymers on pressurization of RT-qPCR premix reagents

[0095] According to the method in Example 1, 17-22 in the fully premixed reaction solution B were prepared, and fully premixed reaction solutions 17 and 18 were used as the fourth group, fully premixed reaction solutions 17, 19 and 20 were used as the fifth group, and fully premixed reaction solutions 20-22 were used as the sixth group. Each of the above three groups was divided into two parts, one of which was stored at -20°C for 7 days as a control group, and the other was stored at 37°C for 7 days as a pressurized experimental group. After the pressurization, 10 pg of total RNA was added, and the experimental reaction was carried out according to Table 3. Two replicates were performed for each system, and M-54 and M-5 genes were tested respectively to detect the effects of different single-stranded DNA binding protein monomers and polymers on the pressurized reaction of the one-step RT-qPCR fully premixed reagent.

[0096] Experimental results analysis:

[0097] The experimental results are shown in Figures 3A-3C ,in Figure 3A These are the experimental results of Cspa dimer and monomer in the pressurized reaction of RT-qPCR full premix reagent. Figure 3B The experimental results of Cspa-T4GP32 dimer and monomer in the pressurized reaction of RT-qPCR premix reagent are shown in Figure 2. Figure 3C The experimental results of T4GP32-Ecoli-SSB-T4GP32 trimer and monomer in the RT-qPCR full premix reagent pressurization reaction. Figures 3A-3C It can be seen that the full premix reagent containing heteropolymers can maintain the stability of the full premix reagent under pressure more significantly than the full premix reagent containing the corresponding single-stranded DNA binding protein monomer. After adding Cspa monomer to the reaction, the full premix working solution of the mixed primer probe only retains 45% to 60% of its amplification platform at -20°C after pressurization, while the full premix working solution with the addition of Cspa dimer still retains more than 90% of its amplification platform at -20°C after pressurization, which is about 40% higher than the retention value of the platform when adding Cspa monomer. After adding Cspa-T4GP32 dimer or T4GP32-Ecoli-SSB-T4GP32 trimer to the amplification system, the full premix working solution of the mixed primer probe can still retain more than 95% of its amplification platform at -20°C after pressurization. Compared with the full premix reagent with only monomer added, the retention value of the amplification platform of the full premix reagent containing polymers after pressurization is increased by more than 30%. It can be seen that adding single-stranded DNA binding protein polymers to the full premix reagent can significantly improve the stability of the amplification platform of the full premix working solution after pressurization at 37°C for 7 days.

[0098] Example 5: Effect of single-stranded DNA binding protein polymers on qPCR reactions

[0099] According to the method in Example 1, 23-29 in the fully premixed reaction solution C were prepared, and 10 pg of extracted DNA and corresponding primer probes were added to prepare single reaction systems 23-29. Each system was repeated twice to test H-02, H-17 and M-13 genes respectively, and the effect of single-stranded DNA binding protein polymers on DNA qPCR reaction in the same reaction was detected.

[0100] The prepared reaction system was reacted according to Table 6.

[0101] Table 6: Reaction Procedure

[0102]

[0103] Experimental results analysis:

[0104] The experimental results are shown in Figures 4A-4C ,in Figure 4A The results of qPCR detection of different single-stranded DNA binding protein monomers and polymers in the H-02 system. Figure 4BThe results of qPCR detection of monomers and polymers of different single-stranded DNA binding proteins in the H-17 system. Figure 4C The results of qPCR detection of monomers and polymers of different single-stranded DNA binding proteins in the M-51 system. Figures 4A-4C It can be seen that compared with the control group with only single-stranded DNA binding protein monomers, the amplification platform of the reagent was improved by 50% to 450% after adding different single-stranded DNA binding protein polymers to the qPCR reaction system. In the H-02 gene detection, compared with the addition of single-stranded DNA binding protein monomers, the improvement of the amplification platform by different polymers was between 20% and 60%; in the H17 gene detection, the improvement of the amplification platform was more significant, compared with the control group with only single-stranded DNA binding protein monomers, the improvement of the amplification platform by different polymers was between 150% and 435%; in the detection of mouse gene M-51, compared with the addition of single-stranded DNA binding protein monomers, the system with added polymers also showed a significant improvement in the amplification platform, ranging from 20% to 130%. It can be seen that adding single-stranded DNA binding protein polymers to the DNA qPCR reaction system can also improve the amplification platform of the amplification reagent.

[0105] Example 6: Effect of single-stranded DNA binding protein polymers on pressurization of qPCR premix reagents

[0106] According to the method in Example 1, 30-33 in the fully premixed reaction solution D were prepared, and fully premixed reaction solutions 30 and 31 were the seventh group, and fully premixed reaction solutions 31-33 were the eighth group. Each of the above two groups of reagents was divided into two parts, one was stored at -20°C for 7 days as a control group, and the other was stored at 37°C for 7 days as a pressurized experimental group. After the pressurization, 10 pg of mouse DNA was added, and the experimental reaction was carried out according to Table 5. Two replicates were performed for each system, and the M-15 and M-18 genes were tested respectively to detect the effects of different single-stranded DNA binding protein monomers and polymers on the pressurized reaction of the DNAqPCR fully premixed reagent.

[0107] Experimental results analysis:

[0108] The experimental results are shown in Figure 5A-5B ,in Figure 5A These are the experimental results of Cspa dimer and monomer in the pressurized reaction of the qPCR full premix reagent. Figure 5B The experimental results of Cspa-T4GP32 dimer and monomer in the pressure reaction of qPCR premix reagent. Figure 5A-5BIt can be seen that compared with the full premix reagent in which only single-stranded DNA binding protein monomers are added to the qPCR full premix reagent, the full premix reagent containing different forms of single-stranded DNA binding protein polymers can more significantly maintain the stability of the full premix reagent under pressure. The full premix working solution containing Cspa monomers only retains 50% to 60% of the amplification platform height at -20°C after pressurization, while the full premix working solution containing Cspa dimers still retains more than 90% of the amplification platform height at -20°C after pressurization, which is about 40% higher than the amplification platform retention value of the full premix reagent containing Cspa monomers. The full premix working solution containing Cspa-T4GP32 dimers can still retain more than 95% of the amplification platform at -20°C after pressurization. Compared with the full premix reagent containing only monomers, the amplification platform retention value of the full premix reagent containing polymers after pressurization is increased by 30% to 60%. It can be seen that adding single-stranded DNA binding protein polymers to the qPCR full premix reagent can significantly improve the stability of the amplification platform of the qPCR full premix working solution after pressurization at 37°C for 7 days.

[0109] Example 7: Effects of single-stranded DNA binding protein polymers on RT-qPCR reagents in different ionic buffers

[0110] 34-48 in the fully premixed reaction solution E was prepared according to the method in Example 1, and 10 pg of the extracted total RNA and the corresponding primer probe were added to prepare a single reaction system 34-48. Two replicates were made for each system, and the reaction was carried out according to Table 5 to test the M-51 gene. Reaction systems 34-38, 39-43, and 44-48 were used as a group to verify the effects of single-stranded DNA binding protein monomers and polymers on one-step RT-qPCR reactions in different ion buffers.

[0111] The experimental results are shown in 6A-6C, where Fig. 6A The RT-qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 1 are shown in Figure 1. Figure 6B The RT-qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 4 are shown in Figure 4. Figure 6C The RT-qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 5. Figures 6A-6CIt can be seen that in the RT-qPCR reaction solution composed of different ion components, compared with the control group without adding single-stranded binding protein, after adding Cspa and T4GP32 monomers to the reaction system, the amplification platform of the reagent increased by 20% to 90%, and the reagent amplification platform can be increased by 80% to 380% by adding single-stranded DNA binding protein polymers to the reaction system. The reagent amplification platform of adding polymers is increased by 40% to 150% compared with the reagent amplification platform of adding corresponding monomers. Especially in buffer 5, the amplification platform of adding Cspa dimer is increased by about 144% compared with the amplification platform of adding Cspa monomer; and the addition of Cspa-T4GP32 dimer is increased by 120% to 150% compared with the addition of Cspa monomer and T4GP32 monomer. It can be seen that the addition of single-stranded binding protein polymers to RT-qPCR reaction solutions with different ion compositions can significantly improve the amplification platform of the reaction reagent, and the effect is more significant than that of the corresponding single-stranded binding protein monomers.

[0112] Example 8: Effects of single-stranded DNA binding protein polymers on qPCR reagents in different ionic buffers

[0113] 49-63 in the fully premixed reaction solution F was prepared according to the method in Example 1, and 10 pg of the extracted DNA and the corresponding primer probe were added to prepare single reaction systems 49-63. Two replicates were made for each system, and the reaction was carried out according to Table 6 to test the H-17 gene. Reaction systems 49-53, 54-58, and 59-63 were used as a group to verify the effects of single-stranded DNA binding protein monomers and polymers on qPCR reactions in different ion buffers.

[0114] The experimental results are shown in 7A-7C, where Fig. 7A The qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 1 are shown in Figure 1. Figure 7B The qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 2 are shown in Figure 2. Figure 7C The qPCR results of single-stranded DNA binding protein monomers and polymers in buffer 3 are shown in Table 3. Figures 6A-6CIt can be seen that in the qPCR reaction solution composed of different ion components, compared with the control group without adding single-stranded binding protein, after adding Cspa and T4GP32 monomers to the reaction system, the amplification platform of the reagent increased by 30% to 100%. When different polymers were added to the reaction system, the amplification platform of the reagent could be increased by 150% to 400%. The amplification platform of the reagent with the addition of polymers was increased by 60% to 200% compared with the reagent with the addition of the corresponding monomer. In particular, in buffer 2, the amplification platform of the reagent with the addition of Cspa dimer was increased by about 180% compared with the reagent with the addition of Cspa monomer, and the amplification platform of the reagent with the addition of Cspa-T4GP32 dimer was increased by 100% to 140% compared with the reagent with the addition of Cspa monomer and T4GP32 monomer. It can be seen that the improvement of the amplification platform of the qPCR reagent by adding single-stranded DNA binding protein polymers does not depend on a specific ion component and has universal applicability.

[0115] Protein sequence information

[0116] Cspa monomer SEQ ID NO:34

[0117] SGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

[0118] T4GP32 monomer SEQ ID NO:35

[0119] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAM IAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL

[0120] Ecoli-SSB monomer SEQ ID NO:36

[0121] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF

[0122] Cspa dimer SEQ ID NO:37

[0123] SGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSLRKLVRHKRVHSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

[0124] T4GP32 dimer SEQ ID NO:38

[0125] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLRKLVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL

[0126] Ecoli-SSB dimer SEQ ID NO:39

[0127] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFHLVKDARGHKMASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF

[0128] Cspa-T4GP32 Dimer SEQ ID NO:40

[0129] SGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSLRKLVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQ

[0130] SAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDL

[0131] DAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL

[0132] Cspa Triplet SEQ ID NO:41

[0133] SGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

[0134] RKLVRHKRVHSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAK

[0135] GPAAGNVTSLRKLVRHKRVHSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQ

[0136] KVSFTIESGAKGPAAGNVTSL

[0137] T4GP32 triplet SEQ ID NO:42

[0138] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKK

[0139] NGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKY

[0140] RFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQ

[0141] KELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTK

[0142] TEDDFMSSSSGSSSSADDTDLDDLLNDLRKLVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGE

[0143] WKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLY

[0144] NTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCP

[0145] WEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFG

[0146] QVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLRK

[0147] LVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAI

[0148] LVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPE

[0149] NEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAI

[0150] PNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAF

[0151] NVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL

[0152] E. coli - SSB trimer SEQ ID NO:43

[0153] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVA

[0154] SEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQG

[0155] GWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFHLVKDARGHKMASRGVNKVILVGN

[0156] LGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEG

[0157] QLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQ

[0158] FSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFHLVKDARGHKMASRGVNKVILVGNLGQDPEVRYMPNG

[0159] GAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQ

[0160] DRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAP

[0161] AAPSNEPPMDFDDDIPF

[0162] Cspa-T4GP32-Ecoli-SSB triple sequence SEQ ID NO:44

[0163] SGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

[0164] RKLVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAP

[0165] FAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPA

[0166] APENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQ

[0167] SAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDL

[0168] DAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLHLVKDARGHKMASRGVNKVILVGNLGQDP

[0169] EVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTR

[0170] KWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF

[0171] T4GP32 - Cspa - Ecoli - SSB triple - body SEQ ID NO:45

[0172] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLHLVKDARGHKSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSLRKLVRHKRVHMASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF

[0173] Ecoli-SSB-T4GP32-Cspa triple sequence SEQ ID NO:46

[0174] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFRKLVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLHLVKDARGHKSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

[0175] T4 GP32-Ecoli-SSB-T4 GP32 triple sequence SEQ ID NO:47

[0176] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLHLVKDARGHKMASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFRKLVRHKRVHMFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL

[0177] Cspa tetramer SEQ ID NO:48

[0178] SGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSLRKLVRHKRVHSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSLRKLVRHKRVHSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSLRKLVRHKRVHSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL

[0179] T4GP32 tetramer SEQ ID NO:49

[0180]

[0181] Ecoli-SSB quadruplex SEQ ID NO:50

[0182] MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFHLVKDARGHKMASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFHLVKDARGHKMASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPFHLVKDARGHKMASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKATGEMKEQTEWHRVVLFGKLAEVASEYLRKGSQVYIEGQLRTRKWTDQSGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGNIGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDIPF。

Claims

1. A mixture comprising a DNA polymerase, a buffer substance, a metal salt and a single-stranded DNA binding protein polymer.

2. The mixture as claimed in claim 1, wherein the single-stranded DNA binding protein polymer comprises at least two single-stranded DNA binding protein monomers; preferably, the single-stranded DNA binding protein polymer is a dimer, trimer or tetramer.

3. The mixture as claimed in claim 2, wherein the single-stranded DNA binding protein monomers in the single-stranded DNA binding protein polymer are of the same type or different types.

4. The mixture according to claim 3, wherein the single-stranded DNA binding protein monomer comprises Escherichia coli single-stranded binding protein, Thermus thermophilus single-stranded binding protein, Methanococcus jannaschii single-stranded binding protein, T4GP32 protein, Cspa cold-sensitive protein, Muts protein and / or RecA protein.

5. The mixture according to claim 3, wherein the single-stranded DNA binding protein polymer comprises Escherichia coli single-stranded binding protein, T4GP32 protein and / or Cspa cold-sensitive protein; preferably, the single-stranded DNA binding protein polymer is Cspa cold-sensitive protein polymer; preferably, the single-stranded DNA binding protein polymer is Escherichia coli single-stranded binding protein polymer; preferably, the single-stranded DNA binding protein polymer is T4GP32 protein polymer; preferably, the single-stranded DNA binding protein polymer is Cspa cold-sensitive protein and T4GP32 protein polymer; preferably, the single-stranded DNA binding protein polymer is T4GP32 protein and Escherichia coli single-stranded binding protein polymer; preferably, the single-stranded DNA binding protein polymer is Cspa cold-sensitive protein, T4GP32 protein and Escherichia coli single-stranded binding protein polymer.

6. The mixture according to claim 1, wherein the single-stranded DNA binding protein polymer comprises any one or more of the proteins shown in SEQ ID NOs: 37-50.

7. The mixture as claimed in claim 2, wherein the single-stranded DNA binding protein dimer comprises any one or more of the proteins shown in SEQ ID NOs: 37-40, the single-stranded DNA binding protein trimer comprises any one or more of the proteins shown in SEQ ID NOs: 41-47, and the single-stranded DNA binding protein tetramer comprises any one or more of the proteins shown in SEQ ID NOs: 48-50.

8. The mixture as claimed in claim 1, wherein the molar ratio of the single-stranded DNA binding protein polymer to the single-stranded DNA in a single reaction is 0.5-20; preferably 1-10; more preferably 2-5.

9. The mixture as claimed in claim 1, wherein the single-stranded DNA binding protein polymer is obtained by direct expression in bacteria; preferably, the single-stranded DNA binding protein polymer is obtained by expression in Escherichia coli.

10. The mixture according to claim 1, wherein the single-stranded DNA binding protein polymer is obtained by in vitro cross-linking polymerization of single-stranded DNA binding protein monomers.

11. The mixture as claimed in claim 1, wherein there is a connecting portion between the monomers in the single-stranded DNA binding protein polymer, and the connecting portion consists of 1-25 arbitrary amino acid sequences; preferably, the connecting portion consists of 5-20 arbitrary amino acid sequences; preferably, the connecting portion consists of 8-15 arbitrary amino acid sequences.

12. The mixture of claim 1, wherein the DNA polymerase comprises a DNA-dependent DNA polymerase and / or an RNA-dependent DNA polymerase.

13. The mixture of claim 12, wherein the DNA-dependent DNA polymerase comprises one or more of Taq DNA polymerase, Pfu DNA polymerase, and Bst DNA polymerase, and the RNA-dependent DNA polymerase comprises one or more of MMLV reverse transcriptase, AMV reverse transcriptase, and HIV reverse transcriptase.

14. The mixture as claimed in claim 1, wherein the buffer substance comprises at least one of Tris, Hepes, Pipes, Taps or Tricine, and the metal salt comprises one or more of magnesium salt, potassium salt, manganese salt, sodium salt, calcium salt or ammonium salt; preferably, the buffer substance is Tris, and the metal salt comprises magnesium salt and / or potassium salt.

15. The mixture of claim 1, further comprising deoxyribonucleoside triphosphates, wherein the deoxyribonucleoside triphosphates comprise one or more of dATP, dCTP, dTTP, dGTP, and dUTP.

16. The mixture of claim 15, wherein the deoxyribonucleoside triphosphates are a mixture of dATP, dCTP, dTTP, and dGTP; optionally, the deoxyribonucleoside triphosphates further comprise dUTP.

17. The mixture according to claim 1, further comprising a primer, a probe or a fluorescent dye; preferably, the probe is a TaqMan probe.

18. The mixture according to claim 17, wherein the fluorescent dye comprises one of SYBR Green I, LC Green PLUS or EvaGreen; preferably, the fluorescent dye is SYBR Green I.

19. The mixture of any one of claims 1 to 18, further comprising uracil-DNA glycosylase.

20. The mixture according to claim 18, further comprising a template, wherein the template is DNA and / or RNA.

21. A single-stranded DNA binding protein polymer, comprising Escherichia coli single-stranded DNA binding protein, T4GP32 protein and / or Cspa cold-sensitive protein.

22. The single-stranded DNA binding protein multimer according to claim 21, wherein the monomers in the single-stranded DNA binding protein multimer are of the same type; preferably, the single-stranded DNA binding protein multimer is a Cspa cold-sensitive protein multimer, an Escherichia coli single-stranded binding protein multimer or a T4GP32 protein multimer; preferably, the single-stranded DNA binding protein multimer comprises any one or more of the proteins shown in SEQ ID NOs: 37-39, SEQ ID NOs: 41-43, and SEQ ID NOs: 48-50.

23. The single-stranded DNA binding protein multimer according to claim 21, wherein the monomers in the single-stranded DNA binding protein multimer are of different types; preferably, the single-stranded DNA binding protein multimer is a Cspa cold-sensitive protein and T4GP32 protein multimer, a T4GP32 protein and Escherichia coli single-stranded binding protein multimer, a Cspa cold-sensitive protein and T4GP32 protein and Escherichia coli single-stranded binding protein multimer; preferably, the single-stranded DNA binding protein multimer comprises any one or more of the proteins shown in SEQ ID NO: 40, SEQ ID NO: 44-46.

24. A polymerase chain amplification reaction method, comprising the step of adding the single-stranded DNA binding protein polymer according to any one of claims 21 to 23 to a reaction system.

25. The method of claim 24, wherein the molar ratio of the single-stranded DNA binding protein polymer to the single-stranded DNA in a single reaction is 0.5-20; preferably 1-10; and more preferably 2-5.

26. A kit comprising components capable of constituting the mixture according to any one of claims 1 to 20.

27. A kit comprising the single-stranded DNA binding protein multimer according to any one of claims 21 to 23.

Images