A Bst DNA polymerase domain, mutants and their applications

Abstract

The application provides a Bst DNA polymerase domain, a mutant and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the Bst DNA polymerase domain is shown in SEQ ID NO:1. The Bst DNA polymerase mutant is obtained by replacing the natural DNA binding domain of the wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO:1. The Bst DNA polymerase mutant exhibits extremely high catalytic efficiency on dUTP, and also has excellent hot-start characteristics, and can reduce the false positive rate of LAMP technology to close to zero. The Bst DNA polymerase mutant also has extremely strong complex sample tolerance, and the detection sensitivity of the pathogen is 20 times higher than that of the wild-type Bst enzyme, and can meet the early and accurate diagnosis requirements of trace infection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a Bst DNA polymerase domain, a mutant, and its applications. Background Technology

[0002] Loop-mediated isothermal amplification (LAMP) technology has shown great potential for rapid on-site detection of pathogens due to its simplicity, speed, and efficiency. However, its practical application, especially in the detection of complex samples in aquaculture and animal husbandry, still faces two major technical bottlenecks: First, there is the issue of false positives caused by aerosol contamination of nucleic acid amplification products. This is a key factor restricting the reliability and widespread adoption of LAMP technology. In laboratory or field environments, high concentrations of products generated from previous amplification reactions easily form aerosols, contaminating subsequent reaction systems and leading to severe false positive results. Currently, the most common solution in the industry is to use dUTP instead of dTTP for amplification, and to treat the product with uracil DNA glycosylase (UDG) before the next round of amplification to degrade the uracil-containing previous amplification products. However, most natural Bst DNA polymerases have a much lower catalytic efficiency for dUTP than for dTTP, resulting in a significant decrease in amplification efficiency and a sharp drop in detection sensitivity after using dUTP. This makes it difficult to balance contamination prevention with high sensitivity, becoming a long-standing pain point in the industry.

[0003] Second, there is the problem of insufficient inhibitor tolerance and detection sensitivity in complex biological samples. In actual testing, samples are usually animal tissues such as liver and spleen, which contain high concentrations of fat, protein, metabolites, and endogenous inhibitors (such as heparin, EDTA, and ions). Existing technologies for modifying Bst DNA polymerase, such as patents CN202310093220.2 and CN202310385054.3, improve enzyme activity or thermostability through single-point mutations; CN202311847381.2 improves thermostability through fusion proteins; and CN113583996B enhances tolerance to inhibitors such as urea and ethanol by chimeric vHP47 and Sto7d domains. These improvements mostly focus on improving one or several properties of the enzyme in standard buffer systems or simple samples. Although these modifications have achieved some success, they have not effectively solved the core problem that enzyme activity is severely inhibited in actual samples containing high concentrations of inhibitors and high levels of fat and protein, resulting in the inability of detection sensitivity to meet the needs of early, trace infection diagnosis.

[0004] In summary, there is an urgent need in this field for a Bst DNA polymerase that can efficiently utilize dUTPs, achieve reliable contamination prevention without sacrificing amplification sensitivity, tolerate various inhibitors in complex samples, and maintain extremely high detection sensitivity in real-world testing scenarios. Currently, there is a lack of solutions that can fundamentally address both of these issues simultaneously. Summary of the Invention

[0005] In view of this, the object of the present invention is a Bst DNA polymerase domain, a mutant thereof and its application.

[0006] The first objective of this invention is to provide a novel Bst DNA polymerase domain for screening, which, when used to replace the native DNA-binding domain of wild-type Bst DNA polymerase, can yield a Bst DNA polymerase mutant.

[0007] The second objective of this invention is to provide a Bst DNA polymerase mutant that exhibits extremely high catalytic efficiency for dUTP, achieving an amplification efficiency of over 80% using dUTP. This Bst DNA polymerase mutant possesses strong tolerance to complex samples and demonstrates excellent resistance to various inhibitors, maintaining high accuracy even when used in the detection of complex samples such as animal liver lysates, which are high in fat and protein. The Bst DNA polymerase mutant exhibits excellent hot-start characteristics, being virtually inactive below 40°C, effectively inhibiting primer dimer formation and non-specific amplification, and is activated only at the reaction temperature, completely eliminating false positives and reducing the false positive rate of LAMP technology to near zero, significantly improving the reliability of detection results. The detection sensitivity for pathogens is as high as 20 copies / μL, 20 times higher than that of wild-type Bst DNA polymerase, meeting the needs for early and accurate diagnosis of trace infections.

[0008] Furthermore, the present invention also provides a screening method for the Bst DNA polymerase mutant, the encoding gene, the recombinant vector, the host cell, a contamination-resistant nucleic acid amplification kit containing the mutant, and its application in rapid detection of complex samples.

[0009] The present invention provides a Bst DNA polymerase domain, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] The present invention provides a Bst DNA polymerase mutant, wherein the Bst DNA polymerase mutant is formed by replacing the natural DNA-binding domain of wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO: 1.

[0011] Preferably, the amino acid sequence of the Bst DNA polymerase mutant is shown in SEQ ID NO: 2.

[0012] The present invention provides a nucleic acid sequence encoding the Bst DNA polymerase mutant, the nucleic acid sequence being shown in SEQ ID NO.3.

[0013] The present invention provides a recombinant vector expressing the Bst DNA polymerase mutant, comprising an initial vector and the nucleic acid sequence thereon.

[0014] The present invention provides a recombinant cell obtained by transferring the recombinant vector into a host cell.

[0015] The present invention provides a nucleic acid amplification kit comprising the Bst DNA polymerase mutant, dUTP, and uracil DNA glycosylation enzyme.

[0016] The present invention provides a detection kit for aquatic pathogens, comprising the Bst DNA polymerase mutant, dUTP, uracil DNA glycosylase, and a set of LAMP primers for aquatic pathogens.

[0017] Preferred aquatic pathogens include shrimp white spot syndrome virus (WSSV), decapod iridovirus DIV1, shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp baculovirus (BP), shrimp yellow head virus (YHV), tiger prawn baculovirus (MBV), shrimp hepatopancreatic parvovirus (HPV), carp herpesvirus type I (CyHV-1), carp herpesvirus type II (CyHV-2), carp edema virus (CEV), koi herpesvirus (KHV), and cytotoxic virus iridovirus (ISK). NV, shrimp hepatocellular carcinoma hepaticulatus EHP, red sea bream iridovirus RSIV, lymphocystis virus LCDV, frog iridovirus LMBV, grouper iridovirus GIV, sturgeon iridovirus AcHV-2, sturgeon herpesvirus type I AcHV-1, sturgeon herpesvirus type II AcHV-2, European eel circovirus EeCV, eel herpesvirus AngHV-1, viral herpesvirus AbHV, Vibrio parahaemolyticus EMS causing acute hepatopancreatic necrosis, Edwardsiella tarda Edwardsiella tarda Fluorescent Pseudomonas Pseudomonas fluorescens Yersinia rumeni Yersinia ruckeri agalactococcus Streptococcus agalactiae Dolphin Streptococcus Streptococcus iniae Pseudomonas putida Pseudomonas putida Nocardia Nocardia and flexorubicin Flexibacter One or more of them.

[0018] Preferably, the test sample of the kit is a sample of liver, spleen or kidney of an aquatic animal; the sample is LAMP amplified using the Bst DNA polymerase mutant at 60-68°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a Bst DNA polymerase domain, the amino acid sequence of which is shown in SEQ ID NO:1. This invention obtains a "hot-start" binding domain through multiple rounds of screening, exhibiting weak binding at low temperatures and strong binding at high temperatures.

[0020] This invention relates to a Bst DNA polymerase mutant obtained by replacing the natural DNA-binding domain of wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO: 1. This mutant achieves highly efficient dUTP synthesis, perfectly balancing contamination prevention and high sensitivity. The Bst DNA polymerase mutant unexpectedly exhibits extremely high catalytic efficiency for dUTP, with amplification efficiency using dUTP exceeding 80% of that using dTTP. This allows for the use of a reliable dUTP / UDG contamination prevention system without sacrificing detection sensitivity, overcoming a long-standing technical bottleneck in the industry.

[0021] The Bst DNA polymerase mutant also possesses excellent hot-start characteristics, completely eliminating false positives: the Bst DNA polymerase mutant is almost inactive below 40°C, effectively inhibiting primer dimer formation and non-specific amplification, and is only activated at the reaction temperature (60~68°C), thereby reducing the false positive rate of LAMP technology to near zero and greatly improving the reliability of the detection results.

[0022] The described Bst DNA polymerase mutant also exhibits extremely strong tolerance to complex samples, resulting in an order-of-magnitude improvement in detection sensitivity: In complex samples such as animal liver lysates, which are high in fat and protein, the Bst DNA polymerase mutant of this invention demonstrates excellent tolerance to multiple inhibitors. Its pathogen detection sensitivity reaches up to 20 copies / μL, which is 20 times higher than that of wild-type Bst enzyme, meeting the needs for early and accurate diagnosis of trace infections.

[0023] Compared to the wild type and other reported mutants, the Bst DNA polymerase mutant of this invention has significant improvements in specific activity, DNA binding efficiency (3-fold increase), and thermal stability, making it an all-around "multi-functional" tool enzyme with excellent overall performance.

[0024] This invention provides a complete set of technical solutions to address industry pain points, from novel structural domains at the source to high-performance mutant products, and then to dedicated anti-contamination reagent kits and application methods, and has extremely high industrial application value. Attached Figure Description

[0025] Figure 1 The above are the amplification curves of the reported Bst DNA polymerase, where 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 represent the amplification curves of different reported mutants, and 11 represents the amplification curve of the commercial enzyme.

[0026] Figure 2 This is a graph showing the relative activity of reported Bst DNA polymerases. In the graph, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 represent the relative activity results of reported Bst DNA polymerases after expression and purification, respectively, and 11 represents the commercial enzyme.

[0027] Figure 3 This represents the improved results of the Bst DNA polymerase in five rounds of EVOLVEpro evolution.

[0028] Figure 4 SDS-PAGE electrophoresis was used to purify proteins from different mutants. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 represent different lanes.

[0029] Figure 5 The graph shows the relative activity of reported Bst DNA polymerases. 0 represents wild type, 1 represents commercial enzyme, and 2, 3, 4, 5, 6, 7, 8, 9, and 10 represent the relative activity detection results of screened Bst DNA polymerases after expression and purification.

[0030] Figure 6 The diagrams show the protein structure and molecular docking. A represents the protein structure, with AlphaFold 3 predicting the three-dimensional structural model of the original Bst DNA polymerase. Different colors and shapes distinguish different domains of the enzyme. B shows the overall structure after the mutant DNA-binding domain is replaced with the Sso7d domain. C shows the binding mode between the DNA-binding domain of the Bst DNA polymerase and the DNA molecule. Molecular docking software simulations demonstrate the interactions between the enzyme and DNA, including hydrogen bonds, electrostatic interactions, and hydrophobic interactions, providing a theoretical basis for enzyme modification and optimization.

[0031] Figure 7The results show the enzyme activity assays at different concentrations of inhibitors. A shows that urea (1-6:0-2M) significantly reduces enzyme activity with increasing urea concentration; Bst DNA polymerase can tolerate 2M urea. B shows that Bst DNA polymerase activity changes with heparin concentration, remaining active at 5U heparin. C shows that enzyme activity decreases with increasing EDTA concentration; the enzyme can tolerate 0.1mM. D shows that enzyme activity decreases with increasing NaCl concentration; the enzyme can tolerate 150mM. E shows that enzyme activity decreases with increasing ethanol concentration; the enzyme can tolerate 15%.

[0032] Figure 8 This image shows a comparison of the protein expression levels of Bst DNA polymerase before and after codon optimization, illustrating the effect of codon optimization on protein expression. In this image, 1 represents the expression level before codon optimization, and 2 and 3 represent the expression levels after codon optimization.

[0033] Figure 9 The results of the sample detection experiment are compared between the purified Bst DNA polymerase and the commercial enzyme in our laboratory. 1 represents the laboratory-purified enzyme; 2, 3, and 4 represent the commercial enzyme. Detailed Implementation

[0034] Specifically, this invention aims to solve the following long-standing technical problems: 1) Solving the problem of balancing contamination prevention and high sensitivity: Existing Bst DNA polymerases have low catalytic efficiency for dUTP, resulting in a significant decrease in nucleic acid amplification efficiency and detection sensitivity when using the dUTP / UDG contamination prevention system. 2) Improving detection performance in complex samples: Existing enzyme mutants are easily affected by inhibitors in high-fat, high-protein real-world samples (such as animal liver tissue), limiting their activity and failing to achieve high-sensitivity detection of early, trace pathogen infections. 3) Eliminating non-specific amplification: Providing a Bst DNA polymerase with excellent hot-start characteristics to completely eliminate the false positive problem caused by room-temperature operation in isothermal amplification techniques such as LAMP.

[0035] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The present invention obtained a novel DNA binding domain through screening and used it to modify Bst DNA polymerase. Unexpectedly, it was found that the resulting mutant showed excellent performance in efficient dUTP synthesis.

[0036] In this invention, the Bst DNA polymerase mutant is formed by replacing the natural DNA-binding domain of the wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of the Bst DNA polymerase mutant is shown in SEQ ID NO: 2, and is as follows: SEQ ID NO: 1 MESPSSEEEKPLAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWALERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGEELAEQLRTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTTGYSTSADVLEKLAPYHEIVENILHYRQL GKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNIS RKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQARLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWYDAK SEQ ID NO:2 MGGGSAEEEEKPLEEMEFAIADEVTEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFFLRPETALASPQFKAWLADETKKKSMFDAKRAIVALKWKGIELRGVAFDLLLAAYLLNPAQDAGDVAAVAKMKQYEAVRSDEAVYGKGAKRAVPDEPVLAEHLVRKAAAAIWALERPFLDELRRNEQDELLTELEQPLAAILAEMFTGVKVD ATVKFKYKGEE KEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKIVENILHYRQLGKLQSTYIEGLLKVVHPDTGKVHTRFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFRRDLDIHTKTAMDIFHVSEDEVTANMRRQAKAVNFGIVYGISAYGLS QNLNITRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEERLQARLLLQVHDELILEAPKEEIERLEKLVPEVMEQAVELRVPLKVDYHYGPTWYDAK The Bst DNA polymerase mutant of this invention has the following characteristics: in a LAMP reaction containing dUTP as the sole source of thymine nucleotides, its amplification efficiency is not less than 80% of the amplification efficiency when dTTP is used as the substrate; it has hot-start characteristics, with its activity not exceeding 10% of its maximum activity below 40°C, and is activated at 60-68°C; it can detect pathogen DNA at a concentration of not less than 20 copies / μL in high-fat, high-protein samples; the mutant is resistant to at least one inhibitor selected from 2M urea, 5U heparin, 0.2mM EDTA, 200mM NaCl, or 15% ethanol.

[0037] The present invention also provides a nucleic acid sequence encoding the Bst DNA polymerase mutant, the nucleic acid sequence being shown in SEQ ID NO.3.

[0038] SEQ ID NO: 3

[0039] ATGGGGGGAGGTTCAGCTGAAGAGGAAAAACCCCTTGAGGAAATGGAATTCGCGATCGCGGACGAAGTAACTGAGGAAATGCTGGCGGACAAAGCAGCCCTGGTTGTTGAAGTTATGGAGGAGAACTATCACGACGCACCGATTGTTGGTATCGCGCTGGTTAATGAACATGGTCGTTTTTTCCTGCGTCCGGAAACCGCATTGGCTAGCCCGCAGTTTAAAGCGTGGCTGGCGGACGAGACGAAGAAAAAATCCATGTTTGATGCGAAACGCGCGATCGTGGCGCTGAAGTGGAAGGGCATCGAGTTACGTGGTGTTGCCTTCGATCTCTTACTCGCCGCGTACCTGCTCAATCCGGCACAGGATGCTGGTGATGTTGCGGCTGTGGCAAAGATGAAACAGTATGAAGCGGTACGCAGCGATGAAGCGGTGTACGGCAAGGGCGCAAAGCGCGCTGTCCCGGACGAGCCCGTGCTGGCGGAGCACCTGGTGAGAAAGGCTGCTGCGATTTGGGCACTGGAGCGCCCGTTTCTGGACGAGCTCCGTCGCAACGAACAGGATGAGTTGCTTACCGAATTGGAACAACCGTTAGCGGCTATTCTGGCTGAGATGGAGTTCACCGGCGTTAAAGTCGATACGAAGCGT GCAACAGTAAAGTTCAAGTACAAGGGAGAAGAGAAGGAAG TAGATATAAGTAAGATAAAGAAGGTATGGAGAGTAGGCAAAATGATAAGTTTCACCTATGATGAGGGTGGAGGAAAG ACTGGTAGAGGAGCTGTAAGCGAGAAAGACGCTCCAAAAGAACTACTACAAATGTTAGAAAAGCAAAAGAAAACATCCTGCACTACCGCCAGCTGGGTAAACTGCAAAGTACCTATATCGAAGGTCTGCTCAAGGTGGTGCACCCGGATACCGGTAAAGTGCACACCCGTTTCAACCAGGCGTTAACCCAGACCGGCCGTCTGTCGTCGACTGAGCCAAATTTGCAAAACATCCCGATTCGTTTGGAAGAGGGCCGTAAAATCCGTCAGGCATTTGTTCCGAGCGAACCGGACTGGCTGATTTTTGCAGCGGACTACTCGCAAATTGAGTTGCGCGTCCTGGCGCATATTGCCGACGACGACAACCTGATTGAGGCCTTTCGTCGCGATTTGGATATTCATACCAAGACGGCAATGGACATCTTTCATGTTAGCGAAGATGAGGTGACGGCCAATATGCGTCGCCAGGCCAAGGCGGTGAACTTTGGTATCGTCTACGGCATTAGCGCGTACGGCCTGTCCCAAAACCTGAACATCACCCGTAAAGAGGCGGCGGAATTCATCGAACGCTACTTCGAGAGCTTCCCGGGTGTGAAACGTTACATGGAGAACATCGTGCAGGAGGCTAAGCAAAAAGGCTATGTTACCACCCTGCTTCACCGCAGACGTTACTTACCGGACATTACCAGCAGAAATTTCAATGTCCGTAGCTTCGCCGAGCGTACCGCAATGAATACCCCGATCCAGGGCAGCGCAGCTGACATCATCAAAAAGGCTATGATTGATCTGGCGGCGCGTCTGAAAGAAGAGCGTTTGCAAGCGCGTTTGTTGTTGCAGGTTCACGATGAGCTGATTTTGGAAGCCCCTAAAGAAGAGATCGAGCGCCTGGAGAAGTTGGTGCCGGAAGTCATGGAGCAAGCTGTGGAGCTGCGCGTTCCGCTGAAGGTGGATTACCACTATGGTCCCGACCTGGTACGACGCCAAG

[0040] The present invention provides a recombinant vector expressing the Bst DNA polymerase mutant, comprising an initial vector and the nucleic acid sequence described above.

[0041] The present invention provides a recombinant cell obtained by transferring the recombinant vector into a host cell.

[0042] This invention provides a nucleic acid amplification kit, comprising the aforementioned Bst DNA polymerase mutant, dUTP, and uracil DNA glycosylation enzyme. In this invention, the nucleic acid amplification kit further comprises an isothermal amplification buffer. This invention does not specifically limit the source of the dUTP and uracil DNA glycosylation enzyme; commercially available products conventional in the art can be used. Preferably, the nucleic acid amplification kit further comprises a nucleic acid release agent; the nucleic acid release agent preferably comprises the following components: 2%–3% sodium deoxycholate, 0.4–0.6 mM PMSF, 10–20 mM EDTA, 0.2%–0.4% urea, and 2–4 mM DTT.

[0043] In this invention, the method of using the nucleic acid amplification kit includes the following steps: a) After mixing, grinding, standing, and centrifuging the sample with the nucleic acid release agent, collect the supernatant as a template; b) Mix and incubate the Bst DNA polymerase mutant, dUTP, primer set, uracil DNA glycosylase and template to degrade any possible uracil-containing contaminants; c) Perform isothermal amplification at 64~66℃ for 40~50 min, and then interpret the results: use real-time fluorescence curves to determine the result; if there is an amplification curve, it is positive, otherwise it is negative; or add SYBR Green I dye after the reaction and observe the color change with the naked eye. If the reaction solution turns green, it is positive, otherwise it is negative.

[0044] The present invention provides a detection kit for aquatic pathogens, comprising the Bst DNA polymerase mutant, dUTP, uracil DNA glycosylase, and a set of LAMP primers for aquatic pathogens.

[0045] The aquatic pathogens mentioned include shrimp white spot syndrome virus (WSSV), decapod iridovirus DIV1, shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp baculovirus (BP), shrimp yellow head virus (YHV), tiger prawn baculovirus (MBV), shrimp hepatopancreatic parvovirus (HPV), carp herpesvirus type I (CyHV-1), carp herpesvirus type II (CyHV-2), carp edema virus (CEV), koi herpesvirus (KHV), and cell edema virus (ISKN). V. Shrimp hepatocellular carcinoma EHP, red sea bream iridovirus RSIV, lymphocystis virus LCDV, frog iridovirus LMBV, grouper iridovirus GIV, sturgeon iridovirus AcHV-2, sturgeon herpesvirus type I AcHV-1, sturgeon herpesvirus type II AcHV-2, European eel circovirus EeCV, eel herpesvirus AngHV-1, viral herpesvirus AbHV, Vibrio parahaemolyticus EMS causing acute hepatopancreatic necrosis, Edwardsiella tarda Edwardsiella tarda Fluorescent Pseudomonas Pseudomonas fluorescens Yersinia rumeni Yersinia ruckeri agalactococcus Streptococcus agalactiae Dolphin Streptococcus Streptococcus iniae Pseudomonas putida Pseudomonas putida Nocardia Nocardia and flexorubicin Flexibacter One or more of them.

[0046] The aquatic pathogen LAMP primer set includes: Primer pairs for white spot syndrome virus (WSSV) in shrimp: F3-WSSV, B3-WSSV, FIP-WSSV, BIP-WSSV, LB-WSSV, and LF-WSSV.

[0047] Primer pairs targeting Decapoda iridovirus (DIV1): F3-DIV1, B3-DIV1, FIP-DIV1, BIP-DIV1, and LF-DIV1.

[0048] Primer pairs targeting infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp: F3-IHHNV, B3-IHHNV, FIP-IHHNV, BIP-IHHNV, and LB-IHHNV.

[0049] Primer pairs for shrimp baculovirus (BP): F3-BP, B3-BP, FIP-BP, BIP-BP, LB-BP, and LF-BP.

[0050] Primer pairs targeting shrimp yellow head virus (YHV): F3-YHV, B3-YHV, FIP-YHV, BIP-YHV, LB-YHV, and LF-YHV.

[0051] Primer pairs targeting baculovirus (MBV) in tiger prawn: F3-MBV, B3-MBV, FIP-MBV, BIP-MBV, LB-MBV, and LF-MBV.

[0052] Primer pairs targeting shrimp hepatopancreatic parvovirus (HPV): F3-HPV, B3-HPV, FIP-HPV, BIP-HPV, LB-HPV, and LF-HPV.

[0053] Primer pairs targeting CyHV-1 (CyHV-1): F3-CyHV1, B3-CyHV1, FIP-CyHV1, BIP-CyHV1, LB-CyHV1, and LF-CyHV1.

[0054] Primer pairs for carp herpesvirus type II (CyHV-2): F3-CyHV2, B3-CyHV2, FIP-CyHV2, BIP-CyHV2, LB-CyHV2, and LF-CyHV2.

[0055] Primer pairs against carp edema virus (CEV): F3-CEV, B3-CEV, FIP-CEV, BIP-CEV, LB-CEV, and LF-CEV.

[0056] Primer pairs against koi herpesvirus (KHV): F3-KHV, B3-KHV, FIP-KHV, BIP-KHV, LB-KHV, and LF-KHV.

[0057] Primer pairs targeting ISKNV (Iridoviruses) of the genus ISKNV: F3-ISKNV, B3-ISKNV, FIP-ISKNV, BIP-ISKNV, LB-ISKNV, and LF-ISKNV.

[0058] Primer pairs targeting Enterocytozoa hepatica (EHP) in shrimp: F3-EHP, B3-EHP, FIP-EHP, BIP-EHP, LB-EHP, and LF-EHP.

[0059] Primer pairs for red sea bream iridovirus (RSIV): F3-RSIV, B3-RSIV, FIP-RSIV, BIP-RSIV, LB-RSIV, and LF-RSIV.

[0060] Primer pairs against lymphocystis virus (LCDV): F3-LCDV, B3-LCDV, FIP-LCDV, BIP-LCDV, LB-LCDV, and LF-LCDV.

[0061] Primer pairs targeting frog iridovirus (LMBV): F3-LMBV, B3-LMBV, FIP-LMBV, BIP-LMBV, LB-LMBV, and LF-LMBV.

[0062] Primer pairs for grouper iridovirus (GIV): F3-GIV, B3-GIV, FIP-GIV, BIP-GIV.

[0063] Primer pairs targeting Sturgeon Iridovirus (WSIV): F3-WSIV, B3-WSIV, FIP-WSIV, BIP-WSIV, and LB-WSIV.

[0064] Primer pairs targeting AciHV-1 (AciHV-1): F3-AciHV1, B3-AciHV1, FIP-AciHV1, BIP-AciHV1, and LF-AciHV1.

[0065] Primer pairs targeting AciHV-2 (AciHV-2): F3-AciHV2, B3-AciHV2, FIP-AciHV2, BIP-AciHV2, and LB-AciHV2.

[0066] Primer pairs for European eel circovirus (EeCV): F3-EeCV, B3-EeCV, FIP-EeCV, BIP-EeCV, and LF-EeCV.

[0067] Primer pairs against anal herpesvirus (AngHV): F3-AngHV, B3-AngHV, FIP-AngHV, BIP-AngHV, and LB-AngHV.

[0068] Primer pairs against viral herpesvirus (AbHV): F3-AbHV, B3-AbHV, FIP-AbHV, BIP-AbHV, LB-AbHV, and LF-AbHV.

[0069] Primer pairs targeting Vibrio parahaemolyticus (EMS), which causes acute hepatopancreatic necrosis: F3-pirB, B3-pirB, FIP-pirB, BIP-pirB, LB-pirB, and LF-pirB.

[0070] Primer pairs for Edwardsiella tarda: F3-ED, B3-ED, FIP-ED, BIP-ED, LB-ED, and LF-ED.

[0071] Primer pairs for Pseudomonas fluorescens: F3-PF, B3-PF, FIP-PF, BIP-PF, LB-PF, and LF-PF.

[0072] Primer pairs against Yersinia rumeni: F3-Yr, B3-Yr, FIP-Yr, BIP-Yr, LB-Yr, and LF-Yr.

[0073] Primer pairs targeting Streptococcus agalactiae: F3-GBS, B3-GBS, FIP-GBS, BIP-GBS, LB-GBS, and LF-GBS.

[0074] Primer pairs targeting Streptococcus dolphinus: F3-Ss, B3-Ss, FIP-Ss, BIP-Ss, and LB-Ss.

[0075] Primer pairs against *Pseudomonas putida*: F3-Psp, B3-Psp, FIP-Psp, BIP-Psp, LB-Psp, and LF-Psp.

[0076] Primer pairs against Nocardia (NOS): F3-NOS, B3-NOS, FIP-NOS, BIP-NOS, LB-NOS, and LF-NOS.

[0077] Primer pairs targeting *Flexobacterium*: F3-Fb, B3-Fb, FIP-Fb, BIP-Fb, LB-Fb, and LF-Fb

[0078] The present invention also provides a method for screening the DNA binding domains, comprising the following steps: a) Construct a library of random peptides, wherein the random peptides are fused with a reporter protein for expression; b) Contact the fusion protein with the immobilized DNA at a first temperature of 4~25℃ to remove the binding material; c) Elute the DNA-bound fusion protein at a second temperature of 60-70°C; d) Repeat steps b) and c) at least 3 times to screen for binding domains that have significantly higher binding strength at high temperatures than at low temperatures; e) Determine the amino acid sequence of the binding domain.

[0079] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0080] Example 1

[0081] Screening for novel DNA-binding domain (NovoDBD-1)

[0082] Construction of a random peptide library: A DNA sequence encoding 75 amino acids was designed, with codons for amino acids 15-65 completely randomized using NNK degenerate codons. This random sequence library was inserted into a modified pET-28a vector using molecular cloning techniques, fusing it with the IgG binding domain of Staphylococcus aureus protein A (for subsequent detection and purification) to construct a plasmid library of the "random peptide-Protein A" fusion protein. The library was transformed into Escherichia coli BL21(DE3) to obtain a library with a size greater than 1×10⁻⁶. 9 A primary strain library.

[0083] Functional filtering: a) Induction of expression and protein preparation: Expand the bacterial strain library and induce expression at 37°C for 3 hours using 0.1 mM IPTG. Collect the bacterial cells, sonicate them, centrifuge to collect the supernatant, and obtain the crude fusion protein extract.

[0084] b) Low-temperature negative selection: The crude extract was co-incubated at 4°C with magnetic beads immobilized with random double-stranded DNA fragments for 1 hour. After thorough washing with pre-cooled buffer, the unbound fraction (flow-through) was collected. This step aims to remove common binding domains of DNA that bind strongly at low temperatures.

[0085] c) High-temperature forward elution: Incubate the above flow-through buffer with fresh immobilized DNA magnetic beads at 65°C for 30 minutes. After washing with pre-cooled buffer, elute with pre-warmed elution buffer at 70°C and collect the eluent. This step aims to capture "hot-start" binding domains that bind weakly at low temperatures and strongly at high temperatures.

[0086] d) Multiple rounds of enrichment: The DNA corresponding to the elution buffer is recovered and amplified, and used as input for the next round of screening. The above "low-temperature negative selection-high-temperature positive selection" process is repeated for 4 rounds, with the high-temperature elution temperature increasing to 65℃ in the first round, 68℃ in the second round, 72℃ in the third round, and 75℃ in the fourth round, in order to gradually apply stronger selection pressure and enrich the domains that have a stronger binding ability to DNA at high temperatures.

[0087] Sequencing: The final enriched DNA library was sequenced, and the sequence with the highest frequency and no significant homology to known protein sequences was selected and named NovoDBD-1. Its amino acid sequence is as SEQ ID NO: 2 shown: MGGGSAEEEKPLEEMEFAIADEVTEEMLADKAALVVEVMEENYHDAPIVGIALVNEHGRFLRPETALASPQFKAWLADETKKKSMFDAKRAIVALKWKGIELR GVAFDLLLAAYLLNPAQDAGDVAAVAKMKQYEAVRSDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWALERPFLDELRRNEQDELLTELEQPLAAILAEMEFTGVKVD ATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQ MLEKQKK IVENILHYRQLGKLQSTYIEGLLKVVHPDTGKVHTRFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIADDDNLIEAFRRDLDIHTKTAMDIFHVSEDEVTANMRRQAKAVNFGIVYGISAYGLS QNLNITRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERTAMNTPIQGSAADIIKKAMIDLAARLKEERLQARLLLQVHDELILEAPKEEIERLEKLVPEVMEQAVELRVPLKVDYHYGPTWYDAK

[0088] Example 2

[0089] Construction, expression and purification of Bst-Novo1 mutant

[0090] Gene synthesis and plasmid construction: Using the wild-type Bst DNA polymerase gene (sequence reference GeneBank: WP_010921102.1) as a template, the sequence encoding the natural DNA binding domain was completely replaced with the NovoDBD-1 sequence obtained in Example 1.

[0091] The obtained Bst DNA polymerase full-length gene was codon optimized using E. coli and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized gene was cloned into the pET-28a(+) vector, and a 6×His tag was introduced at the N-terminus to construct the recombinant expression plasmid pET28a-Bst-Novo1.

[0092] Protein expression: The pET28a-Bst-Novo1 plasmid was transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and cultured at 37°C and 200 rpm until OD600. 600 The concentration was 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the temperature was lowered to 20℃ to induce expression for 16 hours.

[0093] Protein purification: Bacterial cells were collected and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole). After sonication and centrifugation, the supernatant was collected. The supernatant was heat-treated in a 65°C water bath for 30 minutes, followed by centrifugation to remove denatured proteins. The heat-treated supernatant was loaded onto a Ni-NTA affinity chromatography column, washed with buffer containing 50 mM imidazole, and finally eluted with buffer containing 250 mM imidazole. SDS-PAGE analysis revealed a Bst-Novo1 mutant protein with an apparent molecular weight of approximately 68 kDa and a purity higher than 95%.

[0094] Example 3

[0095] Performance verification of the Bst-Novo1 mutant

[0096] 1. dUTP synthesis efficiency detection: Two identical LAMP reaction systems were prepared with the following composition: 1.6 μM M IP primer, 1.6 μM BIP primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 0.4 μM Loop F primer, 0.4 μM Loop B primer, 0.4 mM dNTPs (dUTP group: 0.4 mM each of dATP, dCTP, dGTP, and dUTP; dTTP group: 0.4 mM each of dATP, dCTP, dGTP, and dTTP), 8 U Bst-Novo1 mutant, 1× Isothermal Amplification Buffer (20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Tween 20), 1× EvaGreen fluorescent dye, 1× 10 4 Copies of λ DNA template and sterile ultrapure water were added to a final volume of 25 μL. Equal amounts of the Bst-Novo1 mutant prepared in Example 2, along with DNA template and primers, were added, using dTTP and dUTP (as the sole source of thymine nucleotides) as substrates. Real-time fluorescence amplification was performed at 65°C for 60 minutes, with fluorescence signals acquired every 30 seconds. The amplification curves were recorded, and their Ct values ​​and endpoint fluorescence intensities were compared.

[0097] Experimental results:

[0098] Comparative experiment:

[0099] The results showed that the Bst-Novo1 mutant of this invention achieved 85% of the amplification efficiency of dTTP in the system using dTTP, which was significantly higher than that of wild-type Bst enzyme (about 30%) and tested commercial enzyme.

[0100] 2. Validation of hot-start characteristics: The LAMP reaction system containing the Bst-Novo1 mutant was pretreated at 25℃ and 37℃ for 60 minutes, respectively, and then immediately transferred to 65℃ for amplification, with the untreated reaction as a control. A template-free control (NTC) was also set up to observe non-specific amplification.

[0101] The reaction systems pretreated at 25℃ and 37℃ showed no amplification curves in the NTC wells, while the positive control wells all showed normal S-shaped amplification curves, indicating that Bst-Novo1 has perfect hot-start characteristics and can effectively inhibit non-specific amplification at room temperature.

[0102] 3. Sensitivity test for complex samples: Healthy pig liver tissue was collected and homogenized with PBS at a weight-to-volume ratio of 1g:5ml. The supernatant was then centrifuged and used as the complex sample matrix. A serially diluted WSSV (white spot syndrome virus) standard plasmid (10⁻¹¹ g / v) was then added to this matrix. 6 Up to 10 0 (Copies / μL). LAMP assay was performed using the Bst-Novo1 mutant.

[0103] Results: In porcine liver tissue matrix, Bst-Novo1 achieved a detection limit of 20 copies / μL for WSSV. Under the same conditions, the detection limit of wild-type Bst enzyme was 400 copies / μL, representing a 20-fold increase in sensitivity.

[0104] 4. Inhibitor tolerance test: Methods: Different concentrations of inhibitors (urea, heparin, EDTA, NaCl, ethanol) were added to the standard LAMP reaction system, and amplification was performed using Bst-Novo1. The relative activity was calculated with the reaction activity without inhibitors as 100%.

[0105] The results are as follows Figure 7As shown, the Bst-Novo1 mutant maintained more than 80% of its amplification activity in 2M urea, 5U heparin, 0.2mM EDTA, 200mM NaCl and 15% ethanol, demonstrating excellent inhibitor tolerance.

[0106] Example 4

[0107] Application of anti-contamination nucleic acid detection kits

[0108] Kit Composition: This embodiment provides a contamination-resistant nucleic acid detection kit, comprising: Bst-Novo1 mutant purified in Example 2 (8 U / reaction), dUTP (0.4 mM), uracil DNA glycosylase (UDG, 0.1 U / reaction), 10× isothermal amplification buffer (200 mM Tris-HCl pH 8.8, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% Tween 20), a specific LAMP primer dry powder mixture tube for different aquatic pathogens (containing F3, B3, FIP, BIP primers, and some pathogens include Loop primers). This embodiment uses shrimp pathogens and fish pathogens as examples for verification. Nucleic acid release agent (containing 2% sodium deoxycholate, 0.5 mM PMSF, 15 mM EDTA, 0.3% urea, 3 mM DTT), positive and negative controls.

[0109] Reaction system composition (25 μL system):

[0110] Detection method: a) Sample processing: Shrimp sample: Take about 100 mg of shrimp hepatopancreas tissue, add 500 μL of nucleic acid release agent, grind evenly, let stand at room temperature for 10 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant as template.

[0111] Fish samples: Take about 100 mg of kidney tissue from cyprinid fish, add 500 μL of nucleic acid release agent, grind evenly, let stand at room temperature for 10 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant as template.

[0112] b) UDG pretreatment: Prepare the reaction mixture according to the above reaction system, dispense it into each reaction tube, add the template and UDG, and incubate at 37°C for 10 minutes to degrade any possible uracil-containing contaminants.

[0113] c) Isothermal amplification: Immediately transfer the reaction tube to a 65℃ metal bath or a real-time PCR instrument and react for 45 minutes.

[0114] d) Result interpretation: The result is determined by real-time fluorescence curve. The presence of an amplification curve indicates a positive result, while the absence of an amplification curve indicates a negative result. Alternatively, after the reaction is completed, SYBR Green I dye is added and the color change is observed with the naked eye. If the reaction solution turns green, the result is positive; otherwise, the result is negative.

[0115] Test results: 1. Shrimp sample test results This kit was used to detect various shrimp pathogens in clinically collected shrimp hepatopancreas samples. The results are as follows:

[0116] 2. Fish sample test results

[0117] This kit was used to detect multiple fish pathogens in clinically collected fish kidney samples. The results are as follows:

[0118] Pollution control experiment: To verify the anti-contamination effect of this kit, a contamination control group was set up: no UDG was added to the reaction system, and previously amplified product aerosols were added (simulating a contaminated environment). The results showed:

[0119] Results analysis: Detection sensitivity and specificity: The detection results of 270 shrimp samples with 9 pathogens and 430 fish samples with 22 pathogens showed that the detection results of the kit of this invention had a 100% concordance rate with the qPCR verification results. The Ct values ​​of positive samples were in the range of 21.3 to 29.8, indicating that the kit maintained extremely high detection sensitivity and specificity for a variety of aquatic pathogens in complex tissue samples.

[0120] Contamination prevention effect: The results of the contamination control group showed that the group without UDG added had significant amplification (Ct value of about 28-29) in the presence of aerosol contamination of previous amplification products, while the experimental groups with UDG added had no non-specific amplification. This indicates that the UDG pretreatment step of this kit can effectively degrade previous amplification products containing uracil and prevent false positive results.

[0121] Applicability to different sample types: Stable and reliable test results were obtained for both shrimp hepatopancreas tissue (high-fat sample) and fish kidney tissue (high-protein sample), demonstrating that this kit has good tolerance to complex biological samples from different sources.

[0122] The above results demonstrate that the anti-contamination nucleic acid detection kit provided by this invention can effectively prevent contamination by previous laboratory amplification products, while maintaining high sensitivity and specificity for a variety of aquatic pathogens in complex tissue samples, meeting the needs for rapid on-site detection of aquatic pathogens.

[0123] Table 1 Primer sequences of aquatic pathogens

[0124] As demonstrated by the above embodiments, compared to the wild type and other reported mutants, the Bst DNA polymerase mutant of this invention exhibits significant improvements in specific activity, DNA binding efficiency (3-fold increase), and thermostability, making it a versatile tool enzyme with excellent overall performance. From its novel structural domain at its source, to the high-performance mutant product, and to the dedicated anti-contamination kit and application methods, this invention provides a complete technical solution to address industry pain points, possessing extremely high industrial application value.

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Bst DNA polymerase domain, characterized in that, The amino acid sequence of the Bst DNA polymerase domain is shown in SEQ ID NO:

1.

2. A Bst DNA polymerase mutant, characterized in that, The Bst DNA polymerase mutant is formed by replacing the natural DNA-binding domain of the wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO:

1.

3. The Bst DNA polymerase mutant according to claim 2, characterized in that, The amino acid sequence of the Bst DNA polymerase mutant is shown in SEQ ID NO:

2.

4. A nucleic acid sequence encoding the Bst DNA polymerase mutant of claim 2 or 3, characterized in that, The nucleic acid sequence is shown in SEQ ID NO.

3.

5. A recombinant vector expressing the Bst DNA polymerase mutant of claim 2 or 3, characterized in that, Includes the initial vector and the nucleic acid sequence as described in claim 1.

6. A recombinant cell, characterized in that, It is obtained by transferring the recombinant vector of claim 5 into host cells.

7. A nucleic acid amplification kit, characterized in that, Includes the Bst DNA polymerase mutant, dUTP, and uracil DNA glycosylation enzyme as described in claim 2 or 3.

8. A reagent kit for detecting aquatic pathogens, characterized in that, Includes the Bst DNA polymerase mutant, dUTP, uracil DNA glycosylase, and aquatic pathogen LAMP primer set as described in claim 2 or 3.

9. The reagent kit according to claim 8, characterized in that, Aquatic pathogens include shrimp white spot syndrome virus (WSSV), decapod iridovirus DIV1, shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp baculovirus (BP), shrimp yellow head virus (YHV), tiger prawn baculovirus (MBV), shrimp hepatopancreatic parvovirus (HPV), carp herpesvirus type I (CyHV-1), carp herpesvirus type II (CyHV-2), carp edema virus (CEV), koi herpesvirus (KHV), and cytotoxic virus iridovirus (ISKNV). Enterocytozoon hepatisimidae (EHP) in shrimp, RSIV in red sea bream, LCDV in lymphocystis virus, LMBV in frogs, GIV in grouper, AcHV-2 in sturgeon, AcHV-1 and AcHV-2 in sturgeon, EeCV in European eels, AngHV-1 in eels, AbHV in viral herpesviruses, EMS in Vibrio parahaemolyticus causing acute hepatopancreatic necrosis, and Edwardsiella tarda. Edwardsiella tarda Fluorescent Pseudomonas Pseudomonas fluorescens Yersinia rumeni Yersinia ruckeri agalactococcus Streptococcus agalactiae Dolphin Streptococcus Streptococcus iniae Pseudomonas putida Pseudomonas putida Nocardia Nocardia and flexorubicin Flexibacter One or more of them.

10. The kit according to claim 8 or 9, characterized in that, The test samples for the kit are liver, spleen, or kidney samples from aquatic animals; the samples are LAMP amplified using the Bst DNA polymerase mutant at 60-68°C.

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