Improved PCR amplification enzyme, amplification enzyme antibody and buffer composition of all components lyophilized and direct amplification and application thereof

Abstract

This invention belongs to the field of molecular biology technology and discloses an improved PCR amplification enzyme, amplification enzyme antibody, and buffer composition for full-component lyophilization and direct amplification, as well as their applications. This invention provides an enzyme combination comprising Taq enzyme and reverse transcriptase. The Taq enzyme is obtained by site-directed mutagenesis based on wild-type Taq enzyme, exhibiting superior lyophilization tolerance, interference resistance, thermostability, and amplification specificity. The reverse transcriptase is obtained by site-directed mutagenesis based on wild-type reverse transcriptase, exhibiting good lyophilization-reconstitution activity, thermostability, reverse transcription efficiency, and specificity. Using the above enzyme combination to prepare full-component lyophilized and direct PCR amplification reagents can simultaneously solve the pain points of traditional reagents, such as reliance on low-temperature transportation and storage, the need for extraction before amplification, cumbersome operation, and weak interference resistance.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an improved PCR amplification enzyme, amplification enzyme antibody and buffer composition, and their applications, which are lyophilized and directly amplified from all components. Background Technology

[0002] In the field of molecular diagnostics for disease pathogens, polymerase chain reaction (PCR) technology, with its advantages of high specificity, high sensitivity, and rapid detection, has become a core detection method in animal disease prevention and control, pet clinical diagnosis and treatment, and aquaculture disease monitoring. With the large-scale development of animal husbandry, the upgrading of pet medical needs, and the accelerated intensification of aquaculture, the demand for "readily available, convenient, and efficient" PCR testing solutions is becoming increasingly urgent at grassroots veterinary stations, farm sites, pet hospitals, and testing institutions in remote areas. This has highlighted the limitations of traditional liquid PCR reagents, severely restricting the widespread adoption and effective application of molecular diagnostic technology in grassroots settings.

[0003] First, the dependence on low temperatures has become a core barrier to the distribution and application of reagents. Key functional components of traditional liquid PCR reagents, such as wild-type Taq enzymes and the antibodies used for these enzymes, are all bioactive macromolecules. Their spatial conformation and catalytic activity are highly susceptible to temperature changes, requiring a -20°C cold chain for preservation and transportation. This characteristic not only significantly increases reagent distribution costs—cold chain logistics alone accounting for 15%–30% of the total reagent cost—but also frequently leads to reagent inactivation due to cold chain interruptions in remote pastoral areas, mountainous regions, and aquaculture bases where cold chain facilities are inadequate. For example, in emergency testing for major animal diseases such as African swine fever, temperature fluctuations during reagent transportation often result in false negatives, delaying the timely response to the epidemic.

[0004] Secondly, the cumbersome sample pretreatment process leads to low detection efficiency. Conventional PCR reagents have stringent requirements for template nucleic acid purity. Detection requires sample processing using nucleic acid extraction kits, centrifugation columns, or magnetic bead methods to separate and purify nucleic acids before amplification. This process not only requires specialized experimental equipment and operational skills but also takes 1-2 hours, completely failing to meet the demand for "rapid results" in scenarios such as pet emergency care and on-site disease screening in aquatic products. For example, the traditional process for canine parvovirus testing, common in veterinary hospitals, takes more than 3 hours from sample receipt to report issuance, while grassroots farms require "preliminary screening within half an hour" for avian influenza virus testing—a requirement that current technology cannot meet.

[0005] Furthermore, insufficient anti-interference ability and poor component compatibility limit detection performance. The active site of wild-type amplification enzymes easily binds to impurities in samples (such as heme in animal tissues, polysaccharides in feed, and humic acid in aquatic samples), leading to inhibition of enzyme activity. When using crude extract samples directly for amplification, the false negative rate is as high as 20%. At the same time, the ratio of amplification enzyme, antibody, buffer, and protective agent in existing reagents is mostly empirically combined and lacks systematic optimization. After lyophilization, problems such as low enzyme activity recovery rate and poor stability after reconstitution often occur. Even if some products attempt lyophilization, they still need to be stored at 4°C for a short period of time, making it impossible to achieve true room temperature storage and transportation.

[0006] Furthermore, from an industry development perspective, the current trend towards grassroots and on-site testing in the livestock, pet, and aquatic product testing markets is very clear. However, the "low-temperature dependence + complex operation" characteristics of traditional PCR reagents create a sharp contradiction with market demand. Although some domestic and international companies have attempted to develop freeze-dried PCR reagents, most focus on single-component freeze-drying. Even after full-component freeze-drying, problems such as decreased amplification efficiency and reduced detection sensitivity still exist, and the core need for direct amplification is generally not addressed. Therefore, developing a PCR detection reagent that optimizes the performance of key components through genetic modification, achieves synergistic freeze-drying of all components, and combines room-temperature storage and transportation stability with the ability to directly amplify crude samples is not only an inevitable choice to overcome existing technological bottlenecks but also a key support for promoting molecular diagnostic technology for diseases from the laboratory to grassroots fields. This invention is a targeted research and development based on this background. Summary of the Invention

[0007] This invention aims to solve at least one of the technical problems existing in the prior art. An improved PCR amplification detection reagent, which is fully lyophilized and directly amplifiable, simultaneously addresses the pain points of traditional reagents—such as low-temperature dependence, the need for extraction before amplification, cumbersome operation, and weak anti-interference ability—through optimization and modification of key functional components and system optimization.

[0008] The first aspect of the present invention is to provide an enzyme combination.

[0009] A second aspect of the present invention aims to provide biomaterials related to enzyme combinations of the first aspect of the present invention.

[0010] A third aspect of the present invention is to provide a reagent.

[0011] The fourth aspect of this invention is to provide a method for preparing the reagents of the third aspect of this invention.

[0012] The fifth aspect of this invention is to provide a reagent kit.

[0013] The sixth aspect of this invention aims to provide the application of the reagent of the third aspect of this invention or the kit of the fifth aspect of this invention in the detection of a target analyte.

[0014] The seventh aspect of the present invention aims to provide a method for detecting a target substance for non-disease diagnostic purposes.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an enzyme combination comprising Taq enzyme and reverse transcriptase; The amino acid sequence of the Taq enzyme is as follows: A1) SEQ ID NO:3; or A2) The amino acid sequence shown in SEQ ID NO:3 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar function; The amino acid sequence of the reverse transcriptase is as follows: B1) SEQ ID NO:6; or B2) The amino acid sequence shown in SEQ ID NO:6 is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

[0016] The Taq enzyme provided by this invention is based on wild-type Taq enzyme and utilizes a combined scheme of G624P+Q680K+V358K+Y430L, specifically a scheme involving the joint mutation of G624P (replacing glycine with proline), Q680K (replacing glutamine with lysine), V358K (replacing valine with lysine), and Y430L (replacing tyrosine with leucine). This scheme optimizes the enzyme's lyophilization tolerance, interference resistance, and thermostability through gene mutation technology, enabling experiments that allow for the lyophilization of all components and direct sample amplification. The Taq enzyme provided by this invention exhibits stable surface charge, dense hydrophobic residues (L / A / V / I / F), and strong hydrophobic core stacking, resulting in good lyophilization tolerance, thermostability, and amplification specificity. It achieves enzyme tolerance to lyophilization, can resist the interference of inhibitors in complex samples on amplification, and maintains enzyme activity ≥85%~90% in a high humidity environment of 40~60℃ for more than 10 months. The recovery rate of rapid lyophilization and reconstitution at -70℃ is ≥95%, making it suitable for extreme environments such as Southeast Asia.

[0017] The reverse transcriptase provided in this invention is based on wild-type reverse transcriptase. Considering the changes in its surface dehydration, domain separation, and active site structure under lyophilization conditions, and in accordance with the protection principles of reverse transcriptase, the RNA-binding domain (RBD region) undergoes an R55K mutation (replacing arginine Arg with lysine Lys) to improve reverse transcription specificity and shorten the side chain. In the polymerase active domain (PAD region), mutations are performed at H225V (replacing histidine His with valine Val), L231F (replacing leucine Leu with phenylalanine Phe), and T251R (replacing threonine Thr with arginine Arg) to enhance the hydrophobic effect of the polymerase active domain, protecting enzyme activity and enhancing stability. In the auxiliary stabilizing domain (ASD region), a mutation at L631G (replacing leucine Leu with glycine Gly) enhances resistance to dehydration damage and aggregation, improves lyophilization tolerance and thermostability, and adapts to lyophilization processes. Overall, this improves the enzyme's lyophilization-reconstitution activity, thermostability, reverse transcription efficiency, and specificity.

[0018] A second aspect of the invention provides biological materials related to the enzyme combination of the first aspect of the invention; said biological material is any one of a1) to a12): a1) A nucleic acid molecule encoding the enzyme combination of the first aspect of the present invention; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

[0019] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0020] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0021] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0022] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0023] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0024] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0025] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0026] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0027] In some embodiments of the present invention, the nucleic acid molecule is as shown in SEQ ID NO:2 or 5.

[0028] A third aspect of the present invention provides the application of the enzyme combination of the first aspect of the present invention in the preparation of products.

[0029] In some embodiments of the present invention, the product includes reagents or kits.

[0030] In some embodiments of the present invention, the product can be used for PCR amplification.

[0031] A fourth aspect of the present invention provides a reagent comprising the enzyme combination and PCR amplification reaction reagent of the first aspect of the present invention.

[0032] In some embodiments of the present invention, the final concentration of the Taq enzyme in the reagent is 1 U.

[0033] In some embodiments of the present invention, the final concentration of the reverse transcriptase in the reagent is 150 U.

[0034] In some embodiments of the present invention, the PCR amplification reaction reagents include Tris-HCl, potassium glutamate, NH4Cl, MgCl2 and dNTPs.

[0035] In some embodiments of the present invention, the final concentration of Tris-HCl in the reagent is 10 to 30 mM, such as any value or a range formed by any two of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 mM.

[0036] In some embodiments of the present invention, the pH value of the Tris-HCl is 8.0 to 8.5.

[0037] In some embodiments of the present invention, the final concentration of potassium glutamate in the reagent is 20 to 40 mM, such as any value or a range formed by any two of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 mM.

[0038] In some embodiments of the present invention, the final concentration of NH4Cl in the reagent is 5 to 15 mM, such as any value or a range formed by any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mM.

[0039] In some embodiments of the present invention, the final concentration of MgCl2 in the reagent is 1-8 mM, such as any value of 1, 2, 3, 4, 5, 6, 7 or 8 mM or a range formed by any two of them.

[0040] In some embodiments of the present invention, the final concentration of the dNTP in the reagent is 0.1 to 0.5 mM, such as any value of 0.1, 0.2, 0.3, 0.4 or 0.5 mM or a range formed by any two of them.

[0041] In some embodiments of the present invention, the PCR amplification reaction reagents further include primers and probes for detecting the target nucleic acid.

[0042] In some embodiments of the present invention, the final concentration of the primer in the reagent is 0.1 to 0.6 μM, such as any value of 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 μM or a range formed by any two of them.

[0043] In some embodiments of the present invention, the final concentration of the probe in the reagent is 0.1 to 0.5 μM, such as any value of 0.1, 0.2, 0.3, 0.4 or 0.5 μM or a range formed by any two of them.

[0044] The primers and probes mentioned above are selected or designed based on the actual target being detected.

[0045] In some embodiments of the present invention, the reagent further includes thiourea, dodecyl polyoxyethylene ether, sodium acetate, and urea. This is used to promote the release and dissociation of nucleic acids in samples that have been directly amplified without pretreatment.

[0046] In some embodiments of the present invention, the dodecyl polyoxyethylene ether comprises Brij-35.

[0047] In some embodiments of the present invention, the final concentration of the thiourea in the reagent is 1% to 5%, such as any value of 1%, 2%, 3%, 4% or 5% or a range formed by any two of them.

[0048] In some embodiments of the present invention, the final concentration of the dodecyl polyoxyethylene ether in the reagent is 0.1% to 0.5%, such as any value of 0.1%, 0.2%, 0.3%, 0.4% or 0.5% or a range formed by any two of them.

[0049] In some embodiments of the present invention, the final concentration of sodium acetate in the reagent is 0.1% to 0.5%, such as any value or a range formed by any two of 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0050] In some embodiments of the present invention, the final concentration of urea in the reagent is 1% to 5%, such as any value of 1%, 2%, 3%, 4% or 5% or a range formed by any two of them.

[0051] In some embodiments of the present invention, the reagent further includes a lyophilization protectant.

[0052] In some embodiments of the present invention, the freeze-drying protectant includes polyethylene glycol, fatty alcohol polyoxyethylene ether, sucrose, and BSA (bovine serum albumin). These components act as protectants during the freeze-drying process. Simultaneously, polyethylene glycol (PEG4000), fatty alcohol polyoxyethylene ether (AEO3), and sucrose ensure that the reagent forms a dense, non-porous, smooth-surfaced, and neatly edged cake-like structure during freeze-drying, and does not deform during storage and transportation, ensuring the stability of its performance after reconstitution.

[0053] In some embodiments of the present invention, the polyethylene glycol includes PEG4000.

[0054] In some embodiments of the present invention, the fatty alcohol polyoxyethylene ether includes fatty alcohol polyoxyethylene ether AEO3.

[0055] In some embodiments of the present invention, the final concentration of the polyethylene glycol in the reagent is 0.4% to 1%, such as any value or a range formed by any two of 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0056] In some embodiments of the present invention, the final concentration of the fatty alcohol polyoxyethylene ether in the reagent is 0.1% to 0.5%, such as any value or a range formed by any two of 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0057] In some embodiments of the present invention, the final concentration of sucrose in the reagent is 1% to 2%, such as any value or a range formed by any two of 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.8%, or 2%.

[0058] In some embodiments of the present invention, the final concentration of BSA in the reagent is 0.4% to 1%, such as any value or a range formed by any two of 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0059] The reagents provided by this invention optimize the key functional components and system of PCR, and simultaneously solve the pain points of traditional reagents, such as reliance on low-temperature transportation and storage, the need for extraction before amplification, cumbersome operation, and weak anti-interference ability.

[0060] The reagent provided by this invention can directly amplify unpurified crude samples. The modified amplification enzymes (i.e., Taq enzyme and reverse transcriptase) have mutated sites that exclude the binding of impurities in the sample. Simultaneously, the addition of thiourea, dodecyl polyoxyethylene ether, sodium acetate, and urea to the buffer solution chelates ionic impurities in the sample, blocks proteases, reduces non-specific amplification, and ensures the effective detection of low-abundance pathogen nucleic acids in crude samples. This reagent can amplify both DNA and RNA simultaneously, or only DNA or RNA, with no difference in effectiveness. It can be directly applied to the co-detection of animal DNA and RNA viruses, the detection of pet viral infections, the detection of various bacterial pathogens, and the detection of aquatic infected samples, with detection limits reaching 10. 2 The sensitivity is consistent with or better than that of traditional liquid stepwise extraction and detection methods, with a specificity of 100%.

[0061] The reagent of this invention can be a lyophilized powder. Due to its resistance to lyophilization, interference, heat stability, and high specificity, it can be stored and transported at room temperature, enabling direct PCR amplification of disease pathogen samples. This overcomes the pain point of reagents requiring -20℃ low-temperature storage and transportation, significantly reducing storage and transportation costs and conditions, eliminating complex sample pretreatment steps, and significantly improving detection speed. This reagent is compatible with all mainstream real-time PCR amplification instruments, supports the co-detection of DNA and RNA, and is suitable for molecular diagnostic scenarios of various disease pathogens.

[0062] A fifth aspect of the present invention provides a method for preparing the reagent of the fourth aspect of the present invention, comprising the following steps: mixing the components.

[0063] During the reagent preparation process (i.e., the mixing process), the temperature should be kept below 4°C to avoid affecting enzyme activity.

[0064] In some embodiments of the present invention, when the reagent is a lyophilized reagent, the preparation method further includes a lyophilization step.

[0065] In some embodiments of the present invention, the freeze-drying includes the steps of freezing, primary drying, and desorption drying.

[0066] In some embodiments of the present invention, the freezing conditions are: maintaining 2-5°C for 8-15 minutes; and cooling to -50--40°C at a rate of 1-2°C / min.

[0067] In some preferred embodiments of the present invention, the freezing conditions are: maintaining 3-5°C for 8-12 minutes; and cooling to -46-43°C at a rate of 1-2°C / min.

[0068] In some more preferred embodiments of the present invention, the freezing conditions are: maintaining 3-4°C for 8-10 min; and cooling to -46--45°C at a rate of 1-2°C / min.

[0069] In some embodiments of the present invention, the conditions for the first drying are: maintaining a temperature of -50 to -40°C for 160 to 200 min; increasing the temperature to -45 to -35°C at a rate of 0.1 to 1°C / min; and maintaining the temperature of -45 to -35°C for 700 to 750 min.

[0070] In some preferred embodiments of the present invention, the conditions for the first drying are: maintaining a temperature of -48 to -40°C for 160 to 190 min; increasing the temperature to -45 to -42°C at a rate of 0.3 to 0.8°C / min; and maintaining the temperature of -45 to -42°C for 710 to 740 min.

[0071] In some more preferred embodiments of the present invention, the conditions for the first drying are: maintaining a temperature of -46 to -44°C for 170 to 18 min; increasing the temperature to -42 to -40°C at a rate of 0.4 to 0.6°C / min; and maintaining the temperature of -42 to -40°C for 715 to 725 min.

[0072] In some embodiments of the present invention, the desorption drying conditions are as follows: heating at 0.1–1 °C / min to 20–30 °C; maintaining the temperature at 20–30 °C for 200–260 min.

[0073] In some preferred embodiments of the present invention, the desorption drying conditions are: heating at 0.1–0.8 °C / min to 22–28 °C; maintaining the temperature at 22–28 °C for 220–260 min.

[0074] In some more preferred embodiments of the present invention, the desorption drying conditions are: heating at 0.4–0.6 °C / min to 24–26 °C; and maintaining at 24–26 °C for 230–250 min.

[0075] In some embodiments of the present invention, the prepared reagent is pre-frozen at -80°C for 3-4 hours before freeze-drying to ensure complete solidification and prevent volume shrinkage during freeze-drying.

[0076] A sixth aspect of the present invention provides a kit comprising the reagent of the fourth aspect of the present invention, a positive control, and a negative control.

[0077] In some embodiments of the present invention, the kit further includes a reconstitution solvent.

[0078] In some embodiments of the present invention, the resolvent comprises sterilized water (such as DEPC double-distilled water).

[0079] In some embodiments of the present invention, the negative control is a 1xTE buffer comprising 0.2%–0.3% polyethylene glycol (PEG4000), 0.1%–0.15% fatty alcohol polyoxyethylene ether AEO3, and 0.1%–0.3% sucrose.

[0080] In some embodiments of the present invention, the positive control comprises 0.2%–0.3% polyethylene glycol (PEG4000), 0.1%–0.15% fatty alcohol polyoxyethylene ether AEO3, 0.1%–0.3% sucrose, and a 1xTE buffer for the positive plasmid. The positive plasmid is conventionally selected according to the different detection targets.

[0081] A seventh aspect of the present invention provides a method for detecting a target object for non-disease diagnostic purposes, comprising the step of detecting the target object using a reagent from the fourth aspect of the present invention or a kit from the sixth aspect of the present invention.

[0082] In some embodiments of the present invention, the target or the nucleic acid of the target is mixed with the reagents of the fourth aspect of the present invention and then amplified by PCR.

[0083] In some embodiments of the present invention, the PCR amplification procedure can be routinely adjusted according to the different detection targets.

[0084] The beneficial effects of this invention are: This invention provides an enzyme combination comprising Taq enzyme and reverse transcriptase. The Taq enzyme is obtained through site-directed mutagenesis of wild-type Taq enzyme, exhibiting superior lyophilization tolerance, interference resistance, thermostability, and amplification specificity. The reverse transcriptase is derived from wild-type reverse transcriptase through site-directed mutagenesis, demonstrating good lyophilization-reconstitution activity, thermostability, reverse transcription efficiency, and specificity. Using this enzyme combination to prepare fully lyophilized reagents for direct PCR amplification simultaneously addresses the drawbacks of traditional reagents, such as reliance on low-temperature transportation and storage, the need for pre-extraction followed by amplification, cumbersome operation, and weak interference resistance.

[0085] This invention discloses an improved PCR amplification and detection reagent that is fully lyophilized and directly amplifiable. The reagent contains a PCR amplification enzyme optimized through site-directed mutagenesis at the gene level, as well as a dedicated buffer and protectant. Its core innovation lies in the targeted improvement of key components of the liquid PCR reagent, enabling direct lyophilization while simultaneously providing the ability to directly amplify and detect samples.

[0086] Addressing the critical drawback of traditional liquid PCR reagents requiring storage and transportation at -20°C, this invention achieves room-temperature storage and transportation by synergistically optimizing the freeze-drying process of the improved amplification enzyme, amplification enzyme antibody, buffer solution, and protective agent system. This significantly reduces storage and transportation costs and limitations. Furthermore, leveraging the performance upgrades of each component, this reagent can directly perform PCR amplification on samples of infectious disease pathogens, eliminating complex sample pretreatment steps, significantly improving detection speed, and is suitable for universal detection of DNA and RNA nucleic acids from pathogens such as bacteria, viruses, and mycoplasma.

[0087] This invention can be widely applied in the field of molecular diagnostics for various infectious disease pathogens, and it has shown outstanding value in animal disease detection, pet clinical testing and aquatic pathogen detection, providing an efficient and convenient solution for rapid testing at the grassroots level and on-site. Attached Figure Description

[0088] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results are from the CSFV / PRRSV-M whole-component lyophilized direct amplification method in Example 1 of this invention.

[0089] Figure 2 The results of the CSFV / PRRSV-M conventional liquid reagent experiment in Application Example 1 of this invention are shown.

[0090] Figure 3 The results are from the CPV / CDV whole-component lyophilized direct amplification method in Example 2 of this invention.

[0091] Figure 4 The results of the CPV / CDV conventional liquid reagent experiment in Application Example 2 of this invention are shown.

[0092] Figure 5 The results are from the SS-2 / HPS full-component lyophilized direct amplification method in Application Example 3 of this invention.

[0093] Figure 6 The results of the SS-2 / HPS conventional liquid reagent experiment in Application Example 3 of this invention are shown.

[0094] Figure 7 The results are from the WSSV whole-component lyophilized direct amplification method in Application Example 4 of this invention.

[0095] Figure 8 The results of the WSSV conventional liquid reagent experiment in Application Example 4 of this invention are shown. Detailed Implementation

[0096] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0097] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0098] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0099] Example 1: Design and preparation of an improved PCR amplification enzyme (I) Genetic modification of Taq enzyme This embodiment uses wild-type Taq enzyme (GenBank accession number: J04639.1, sequence as shown in SEQ ID NO:1) as a basis and optimizes the enzyme's freeze-drying tolerance, anti-interference and thermal stability through gene mutation technology to achieve the experimental purpose of being able to freeze-dry all components and directly amplify samples.

[0100] Firstly, based on analysis using bioinformatics tools and molecular dynamics software, the core reason for enzyme conformational disruption during freeze-drying is the breakage of hydrogen bonds and separation of structural domains caused by the loss of water molecules. To avoid enzyme domain separation during freeze-drying, the inventors screened various possible mutations and combinations, including G624P (glycine replaced by proline) and / or Q680K (glutamine replaced by lysine) and / or R736D (arginine replaced by aspartic acid) and / or R771K (arginine replaced by lysine).

[0101] Furthermore, in order to achieve a freeze-drying reconstitution activity recovery rate of more than 90%, complete sample testing within 30 minutes, and adapt to emergency disease screening, G624P (glycine Gly replaced with proline Pro) and Q680K (glutamine Gln replaced with lysine Lys) mutations were selected for experiments. The enzymes of this mutation scheme can tolerate freeze-drying and maintain excellent technical parameters.

[0102] Furthermore, the root cause of Taq enzyme's weak anti-interference ability is that the active site easily binds non-specifically to sample impurities (heme, polysaccharides, humic acid, etc.) or is inhibited by metal ions. Mutagenic reagents should consider strengthening the positive charge around the active site to form a charge barrier that repels acidic impurities, weakening the polarity of the inhibitory factor binding site, and reducing the binding of metal ions to polysaccharides. Considered design schemes include V358K (valine replaced by lysine) and / or L367R (leucine replaced by arginine) and / or Y378R (tyrosine replaced by arginine).

[0103] Furthermore, to increase the positive potential around the active site of the mutated Taq enzyme by 20%–30%, reducing the non-specific binding of acidic impurities through electrostatic repulsion while retaining the specific binding site to the nucleic acid template without affecting catalytic efficiency, and achieving a false negative rate of no more than 0.5% for direct amplification of undiluted whole blood samples, with a detection limit of 100 copies / mL for special samples, especially humic acid samples and environmental samples, we selected the V358K (valine replaced by lysine) mutation for experiments. This significantly improved the enzyme's tolerance to interference and enhanced its sensitivity and specificity for amplification of various samples.

[0104] Furthermore, in order to improve the thermostability of the amplification enzyme, strengthen the core rigidity of the enzyme, stabilize the binding domain of the enzyme, promote the construction of surface salt bridges of the enzyme, and improve its high-temperature conformational stability from the inside out, the design schemes considered include Y430L (tyrosine Tyr replaced by leucine Leu), E434K (glutamate Glu replaced by lysine Lys), and S486R (serine Ser replaced by arginine Arg).

[0105] Furthermore, to optimize the high-temperature stability parameters of the enzyme, the inventors selected Y430L (tyrosine Tyr replaced with leucine Leu) for experiments to improve the high-temperature stability of the enzyme.

[0106] Furthermore, based on the requirements of comprehensive sensitivity and specificity of the binding enzyme, including lyophilization tolerance, anti-interference ability, and high-temperature tolerance, analyzed by bioinformatics tools and molecular dynamics software, the inventors finally determined the G624P+Q680K+V358K+Y430L combination scheme, namely, the scheme of joint mutation of G624P (glycine Gly replaced with proline Pro), Q680K (glutamine Gln replaced with lysine Lys), V358K (valine Val replaced with lysine Lys), and Y430L (tyrosine Tyr replaced with leucine Leu), the nucleotide sequence of which is shown in SEQ ID NO:2 and the amino acid sequence is shown in SEQ ID NO:3.

[0107] >J04639.1 Thermus aquaticus DNA polymerase (PolI) gene

[0108] The nucleotide sequence of the modified Taq enzyme:

[0109] Amino acid sequence of the modified Taq enzyme: MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLS K LALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWL L REVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLS P DENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLS K ELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE (SEQ ID NO:3). The bold and underlined parts in this sequence are the modified sites.

[0110] The mutant Taq enzyme has a molecular weight of 92.5 kDa, an isoelectric point of pI=9.2, an instability index of 35.2, a total hydrophobic residue ratio of 42%, and an average hydrophilicity of 0.18. It contains 15% arginine (R) and 15% lysine (K), 15% leucine (L), 11% alanine (A), and 10.4% glutamate (E). It is rich in arginine (R) and lysine (K). The core structure has an α-helix ratio of 65% and a β-sheet ratio of 12% for the catalytic core. The enzyme has stable surface charge, dense hydrophobic residues (L / A / V / I / F), strong hydrophobic core stacking, and good freeze-drying tolerance, thermostability, stability, and amplification specificity.

[0111] This study achieved enzyme tolerance to lyophilization, resistance to interference from inhibitors in complex samples, and maintained enzyme activity ≥85%–90% under high humidity conditions (40–60℃) for over 10 months. It also demonstrated a recovery rate ≥95% after rapid lyophilization and reconstitution at -70℃, making it suitable for extreme environments such as Southeast Asia. This mutated Taq enzyme is designated as the lyophilization-resistant Taq enzyme.

[0112] The above gene (SEQ ID NO:2) was directly synthesized by Shanghai Sangon Biotech Co., Ltd. and ligated into the pET28a expression vector. After receiving the bacterial culture and recombinant plasmid, cloning, expression, and purification were performed as follows: 1. Bacterial culture containing recombinant plasmids with mutant enzymes 1.1 Clean the laminar flow hood with alcohol, irradiate with ultraviolet light for 15 minutes, and strictly follow aseptic procedures to prevent contamination by other microorganisms; turn off the ultraviolet light on the laminar flow hood, turn on the fan and blow for 30 minutes to remove ozone and prevent it from affecting the activity of the bacterial strain; take out the stored bacteria from the -20℃ freezer, record the tube number, dissolve at room temperature, use an inoculation loop to dip a small amount of bacterial solution and streak it on an LB agar plate, and grow overnight at 37℃; 1.2 Pre-amplification: Add 50 mL of 2YT medium to two 250 mL sterilized Erlenmeyer flasks, and add 50 µL of Kan (note that the stock solution concentration of Kan is 30 mg / mL, and the working concentration is 30 µg / mL). Pick fresh single clones and add them to the above Erlenmeyer flasks. Incubate overnight at 37℃ with shaking at 150 rpm. When the OD value is around 2.5, remove the Erlenmeyer flasks and place them in a clean bench for later use. Note that the OD value of the pre-amplification culture should be between 2.0 and 3.0. 1.3 Take six 2 L Erlenmeyer flasks containing 500 mL of sterile 2YT medium, add 500 µL of Kan (Kan stock solution concentration is 30 mg / mL, working concentration is 30 µg / mL) and 15 mL of pre-cultured bacterial solution, and incubate at 37℃ with shaking at 150 rpm for 2.5 h. Measure the OD after incubation. 600When the temperature is between 0.8 and 1.0, remove it and place it on a clean bench for later use. 1.4 Add 0.25 mL of IPTG (note that the IPTG stock solution concentration is 1 M and the working concentration is 0.5 mM), and induce at 37℃ and 150 rpm for 5 h; 1.5 After induction is complete, record the OD at the time of bacterial harvesting. 600 Collect 500 mL of bacterial culture per bottle into a centrifuge bottle, centrifuge at 7000 rpm for 10 min, and discard the supernatant. Record the wet weight of the collected bacteria and sterilize the supernatant.

[0113] 2. Bacterial cell treatment 2.1 Add HS Lysis Buffer (10 mL HS Lysis Buffer per 1 g wet weight) to the above precipitate and mix thoroughly; 2.2 Cell disruption by sonication: The sonication conditions were 350 W, 3 s sonication, 5 s interval, and 20 min sonication (performed in an ice bath). Note that sonication must be complete. When performing SDS-PAGE identification, the sonicated precipitate sample must be retained. The precipitate should be retained before gel analysis. 2.3 After centrifugation at 10000 rpm for 20 min, the supernatant was collected, treated at 75℃ for 20 min, centrifuged at 10000 rpm for 20 min, and the supernatant was filtered through a 0.22 µm filter membrane for purification.

[0114] 3. Purification 3.1 Column packing: Take 5 mL of Ni-NTA and pack it into the purification column. Equilibrate the Ni-NTA column with 5 column volumes of sterile purified water at a flow rate of 1 mL / min. Be sure to stir well before packing to prevent the formation of air bubbles. 3.2 Equilibrate the Ni-NTA column with 5 column volumes of QIAGEN Ni-NTA Spin Kit (31314) Lysis Buffer, then load the sonicated supernatant into the tubing at a flow rate of 0.5 mL / min until OD is reached. 280 Once the value drops to the baseline value (sample is completely injected into the column), retain the wash solution for later detection; 3.3 Use QIAGEN Ni-NTA Spin Kit (31314) Wash Buffer to elute contaminating proteins at a flow rate of 1 mL / min until OD is reached. 280 Once the value drops to the baseline (all contaminating proteins have been eluted), retain the washing buffer for SDS-PAGE analysis. 3.4 The target protein was eluted using the QIAGEN Ni-NTA Spin Kit (31314) elution buffer at a flow rate of 1 mL / min. The main peak was collected when the elution peak appeared, and the OD was recorded.280 Peak value and collection volume to be determined by SDS-PAGE (approximately 2 column volumes). 3.5 Equilibrate the column with 5 column volumes of sterile purified aqueous solution until all impurity peaks are completely eluted. Store the column in 20% ethanol for later use.

[0115] 4. Dialysis and activity assay 4.1 Add the dialysis bag to a boiling water bath for 10 minutes, cool it, and then boil it in a boiling water bath for 10 minutes again. Repeat this process twice. Add the purified HS liquid. Note that when handling the dialysis bag, add a small amount of water to check if it is punctured. The clamps at both ends should be tightened and tied with rubber bands to prevent leakage. 4.2 Place in a dialysis bag and dialyze at 4℃ for 24 hours (change the dialysis solution twice after overnight). 4.3 Transfer the dialyzed enzyme into an ultrafiltration centrifuge tube, centrifuge at 4000 rpm for 20 min, collect the liquid in the upper layer and add Taq enzyme dialysate to rinse the upper layer of the ultrafiltration centrifuge tube (concentration ratio of 10 times), and take out 500 µL to test the activity unit. 4.4 Based on the quality control data for enzyme unit calibration, dilute the enzyme to 50 U / µL and store it at -20℃ for later use.

[0116] (II) Genetic modification of reverse transcriptase (MMLV RT) Furthermore, in inventing a DNA and RNA compatible amplification lyophilization and direct amplification system, the inventors genetically modified the required reverse transcriptase. Based on reverse transcriptase (MMLV RT, GenBank accession number AF033811.1, nucleotide sequence as shown in SEQ ID NO:4), considering its surface dehydration, domain separation, and changes in the structure of the active site under lyophilization conditions, combined with the protection principles of reverse transcriptase, including flexible balance, amino acid composition of the domain linker region, protection of the enzyme active site structure, adaptation of protective agents and enhancers, and synergy of lyophilization processes, the inventors preferentially changed the peripheral sites without directly mutating the catalytic residues.

[0117] Furthermore, using bioinformatics tools and molecular dynamics software, mutation sites were screened, including: 1) RNA binding domain (RBD region): In order to improve the specificity of reverse transcription and make the side chain shorter, an R55K (arginine Arg replaced by lysine Lys) mutation is performed to retain the positive charge, reduce the electrostatic adsorption of non-complementary RNA, reduce non-specific binding by 70%, and decrease the primer dimer formation rate by 60%.

[0118] 2) Polymerase active domain (PAD region): Filling the hydrophobic pocket near the active domain prevents conformational collapse at high temperatures. The H225V mutation (histidine His replaced with valine Val) increases the optimal reverse transcription temperature from 37℃ to 50℃, protecting enzyme activity and enhancing stability. It strengthens the structure around the active domain, improves thermal stability, and does not destroy catalytic activity. The L231F mutation (leucine Leu replaced with phenylalanine Phe) enhances the hydrophobic effect of the polymerase active domain, stabilizes the conformation around the motif, and increases enzyme activity by 20% after incubation at 55℃ for 30 min. The T251R mutation (threonine Thr replaced with arginine Arg) forms an intramolecular salt bridge, stabilizing the dNTP binding site and increasing the activity recovery rate after lyophilization and reconstitution to over 90%.

[0119] 3) The auxiliary stabilizing domain (ASD region) is mutated with L631G (leucine is replaced by glycine), which enhances the resistance to dehydration damage and aggregation, improves freeze-drying tolerance and thermal stability, and adapts to the freeze-drying process. At the same time, it strengthens the hydrophobic effect of the enzyme core region, improves the hydrophilicity of the surface region, reduces freeze-drying aggregation, increases surface flexibility, and avoids mechanical damage during freeze-drying.

[0120] Specifically, the MMLV gene was mutated by R55K+H225V+L231F+T251R+L631G. The nucleotide sequence of the modified MMLV gene is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. This improved the enzyme's lyophilization-reconstitution activity, thermostability, reverse transcription efficiency, and specificity.

[0121] >AF033811.1 Moloney murine leukemia virus, complete genome,8832bp ATGGGACCAATGGGGCAGCCCCTGCAAGTGTTGC TCCAGACACCTCTACCCTCCTC

[0122] Nucleotide sequence of the modified reverse transcriptase: ATGGGACCAATGGGGCAGCCCCTGCAAGTGTTGACCCTAAATATAGAAGATGA GCATCGGCTACATGAGACCTCAAAAGAGCCAGATGTTTCTCTAGGGTCCACATGGCTGTCTGATTTTCCTCAGGCCTGGGCGGAAACCGGGGGCATGGGACTGGCAGTT AAA CAAGCTCCTCTGATCATACCTCTGAAAGCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCACAAGAAGCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCAGGGAATACTGGTACCCTGCCAGTCCCCCTGGAACACGCCCCTGCTACCCGTTAAGAAACCAGGGACTAATGATTATAGGCCTGTCCAGGATCTGAGAGAAGTCAACAAGCGGGTGGAAGACATCCACCCCACCGTGCCCAACCCTTACAACCTCTTGAGCGGGCTCCCACCGTCCCACCAGTGGTACACTGTGCTTGATTTAAAGGATGCCTTTTTCTGCCTGAGACTCCACCCCACCAGTCAGCCTCTCTTCGCCTTTGAGTGGAGAGATCCAGAGATGGGAATCTCAGGACAATTGACCTGGACCAGACTCCCACAGGGTTTCAAAAACAGTCCCACCCTGTTTGATGAGGCACTGCACAGAGACCTAGCAGACTTCCGGATCCAG GTT CCAGACTTGATCCTG TTT CAGTACGTGGATGACTTACTGCTGGCCGCCACTTCTGAGCTAGACTGCCAACAAGGT CG T GG T CTAAAAGCCCTCTTTCTGCCCAAAAGACTTAGCATAATCCATTGTCCAGGACATCAAAAGGGACACAGCGCCGAGGCTAGAGGCAACCGGATGGCTGACCAAGCGGCCCGAAAGGCAGCCATCACAGAGACTCCAGACACCTCTACCCTCCTCtga (SEQ ID NO:5), the underlined and bold part is the mutation site.

[0123] Amino acid sequence of the modified reverse transcriptase: MGPMGQPLQVLTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAV K QAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQ V PDLIL F QYVDDLLLAATSELDCQQG R RALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILA GLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL (SEQ ID NO:6), the underlined and bolded part is the mutation site.

[0124] Furthermore, the MMLV gene with the R55K+H225V+L231F+T251R+L631G mutation was directly synthesized by Shanghai Sangon Biotech Co., Ltd. and ligated into the pET28a expression vector. After receiving the bacterial culture and recombinant plasmid, cloning, expression, and purification were performed as follows: Experimental steps: 1. Bacterial culture containing recombinant plasmids with mutant enzymes 1.1 Clean the laminar flow hood with alcohol, then irradiate with ultraviolet light for 15 minutes. Strict aseptic operation should be performed to prevent contamination by other bacteria. Turn off the ultraviolet light on the laminar flow hood, turn on the fan, and blow for 30 minutes to remove ozone and prevent it from affecting the activity of the bacteria. 1.2 Streak the bacterial culture, remove the culture from the -20℃ freezer, record the tube number, and thaw at room temperature; 1.3 Use an inoculation loop to take a small amount of bacterial suspension and streak it on an LB agar plate, then incubate overnight at 37°C; 1.4 Pre-amplification: Add 50 mL of 2YT medium to two 250 mL Erlenmeyer flasks containing 50 mL of sterilized 2YT medium, and add 50 µL of Kan (Kan stock solution concentration is 30 mg / mL, working concentration is 30 µg / mL); pick fresh single clones and add them to the above Erlenmeyer flasks, and incubate overnight at 37 ℃ with shaking at 150 rpm. When the OD value is around 2.5, remove the Erlenmeyer flasks and place them in a 4 ℃ refrigerator for later use. The OD value of the pre-amplification culture should be between 2.0 and 3.0. 1.5 Take four 2 L Erlenmeyer flasks containing 500 mL of sterilized 2YT medium, add 500 µL of Kan (Kan stock solution concentration is 30 mg / mL, working concentration is 30 µg / mL) and 15 mL of pre-cultured bacterial solution, and incubate at 37℃ with shaking at 150 rpm. 1.6 After culturing for 1.5 h, the OD600 was measured to be between 0.3 and 0.5. The sample was then removed and placed in a clean bench for later use. 1.7 Add 0.1 mL of IPTG (note that the stock solution concentration of IPTG is 1 M and the working concentration is 0.2 mM), and induce overnight at 20 ℃ and 150 rpm; 1.8 After induction is complete, record the OD at the time of bacterial harvest. 600 Collect 500 mL of bacterial culture per bottle into a centrifuge bottle, centrifuge at 7000 rpm for 10 min, and discard the supernatant. Record the wet weight of the collected bacteria and sterilize the supernatant.

[0125] 2. Bacterial cell treatment 2.1 Add RT Lysis Buffer (10 mL RT Lysis Buffer per 1 g wet weight) to the above precipitate and mix thoroughly; 2.2 Cell disruption by sonication: The sonication conditions were 350 W, 3 s sonication, 5 s interval, and 20 min sonication (performed in an ice bath). Note that sonication must be complete. The sonicated precipitate sample must be retained for SDS-PAGE identification. The precipitate should be retained before gel analysis. 2.3 Centrifuge at 10,000 rpm for 20 min, collect the supernatant, filter the supernatant through a 0.22 µm filter membrane, and wait for purification.

[0126] 3. Purification 3.1 Column packing: Take 5 mL of Ni-NTA and pack it into the purification column. Equilibrate the Ni-NTA column with 5 column volumes of sterile purified water at a flow rate of 1 mL / min. Be sure to stir well before packing to prevent the formation of air bubbles. 3.2 Equilibrate the Ni-NTA column with 5 column volumes of QIAGEN Ni-NTA Spin Kit (31314) Lysis Buffer, then load the sonicated supernatant into the tubing at a flow rate of 0.5 mL / min until OD is reached. 280 The value drops to the baseline value (sample completely injected into the column), the wash buffer is retained, and the sample is ready for SDS-PAGE analysis. 3.3 Use the QIAGEN Ni-NTA Spin Kit (31314) Wash Buffe to elute contaminating proteins at a flow rate of 1 mL / min until the OD280 value drops to the baseline value (contaminating proteins are completely eluted). Retain the wash buffer for SDS-PAGE analysis. 3.4 The target protein was eluted using the QIAGEN Ni-NTA Spin Kit (31314) elution buffer at a flow rate of 1 mL / min. The main peak was collected when the elution peak appeared, and the OD was recorded. 280 Peak value and collection volume to be determined by SDS-PAGE (approximately 2 column volumes). 3.5 Equilibrate the column with 5 column volumes of sterile purified aqueous solution until all impurity peaks are completely eluted. Store the column in 20% ethanol for later use.

[0127] 4. Dialysis 4.1 Place the dialysis bag in a boiling water bath for 10 min, cool it, and then boil it again for 10 min. Repeat the boiling water bath twice in total. Add the purified RT liquid. Note that when handling the dialysis bag, add a small amount of water to check if it is punctured. The clamps at both ends should be tightened and tied with rubber bands to prevent leakage. 4.2 Place in a dialysis bag and dialyze at 4℃ for 24 hours (change the dialysis solution twice after overnight).

[0128] 5. Determination of reverse transcriptase concentration and activity Reverse transcriptase with a protein purity of 95% or higher was placed in a clean dialysis bag, sealed at both ends, and placed in a beaker containing 1L of storage buffer. Dialysis was performed overnight at 4°C with slow stirring; this was repeated twice. The reverse transcriptase was collected after dialysis, and its total volume was recorded. The amino acid sequence of the reverse transcriptase was analyzed using software to obtain A. 280 =0.70 mg / mL. Micro-quantitative analysis of enzyme A. 280 The protein concentration was calculated based on the above quantitative relationships, and the required concentration was approximately 18–20 mg / mL. Using total RNA from animal and human peripheral blood lymphocytes as templates, and with currently used reverse transcriptases as references, the activity of the purified reverse transcriptase was determined by quantitative real-time PCR amplification using animal and human internal reference genes as targets.

[0129] The prepared mutant reverse transcriptase was adjusted to a concentration of 200 U / µL according to the calibrated data and stored at -20°C for later use. This mutant reverse transcriptase is designated as the lyophilization-resistant MMLV enzyme.

[0130] Example 2 A PCR lyophilization system (reagent) is prepared from components comprising the following final concentrations: 20 mM Tris-HCl pH 8.3, 30 mM potassium glutamate, 5–10 mM NH4Cl, 2–5 mM MgCl2, primers (different primers are used depending on the target) concentration of 0.2–0.5 μM, probes (different probes are used depending on the target) concentration of 0.1–0.3 μM, 1 U (5 U / µL × 0.2 µL) of lyophilization-resistant Taq enzyme from Example 1, 100–150 U (200 U / µL × 0.5–0.75 µL) of lyophilization-resistant MMLV enzyme from Example 1, and 0.2 mM dNTP Mix (dATP / dCTP / dGTP / dTTP).

[0131] A one-step mixed gradient lyophilization process was used to prepare the PCR lyophilized system (reagents) to ensure the synergistic stability of each component. The specific steps are as follows: (1) Prepare the above PCR lyophilization system under sterile conditions and dispense it into 8-tube PCR tubes at a rate of 50µL / tube. Keep the temperature below 4℃ during the preparation of the above system to avoid affecting enzyme activity. After the reagent is prepared, place it in an ultra-low temperature freezer at -80℃ for 4 hours to allow the reagent to solidify completely and avoid volume shrinkage during lyophilization.

[0132] (2) Gradient drying is carried out using a vacuum freeze dryer according to the steps in Table 1.

[0133] Table 1 Vacuum freeze-drying steps and parameters

[0134] (2) After lyophilization is complete, seal the container, pack it into an aluminum-plastic bag, and seal it with a vacuum packaging machine. The lyophilized PCR reagent can be stored at room temperature.

[0135] Example 3 A PCR lyophilization system (reagent) is prepared from components comprising the following final concentrations: 20 mM Tris-HCl pH 8.3, 30 mM potassium glutamate, 5–10 mM NH4Cl, 2–5 mM MgCl2, primer concentration 0.2–0.5 μM, probe concentration 0.1–0.3 μM, 1 U (5 U / µL × 0.2 µL) of lyophilization-resistant Taq enzyme from Example 1, 100–150 U (200 U / µL × 0.5–0.75 µL) of lyophilization-resistant MMLV enzyme from Example 1, 0.2 mM dNTP Mix (dATP / dCTP / dGTP / dTTP), 1 w / v%–2 w / v% thiourea, 0.1 w / v%–0.5 w / v% dodecyl polyoxyethylene ether (Brij–35), 0.1 w / v% sodium acetate, and 1 w / v%–5 w / v% urea.

[0136] The preparation method of the above PCR lyophilization system (reagent) is the same as in Example 2.

[0137] In the above PCR lyophilization system (reagent), thiourea, dodecyl polyoxyethylene ether (Brij-35), sodium acetate, and urea can be used to promote the release and dissociation of nucleic acids in samples that have been directly amplified without pretreatment.

[0138] Example 4 A PCR lyophilization system (reagent) is prepared from components comprising the following concentrations: 20 mM Tris-HCl pH 8.3, 30 mM potassium glutamate, 5–10 mM NH4Cl, 2–5 mM MgCl2, primer concentration 0.2–0.5 μM, probe concentration 0.1–0.3 μM, 2 U (5 U / µL × 0.2 µL) of lyophilization-resistant Taq enzyme from Example 1, 100–150 U (200 U / µL × 0.5–0.75 µL) of lyophilization-resistant MMLV enzyme from Example 1, and 0.2 mM dNTPs. Mix (dATP / dCTP / dGTP / dTTP), 0.5 w / v%–0.8 w / v% polyethylene glycol (PEG4000), 0.2 w / v%–0.3 w / v% fatty alcohol polyoxyethylene ether (AEO3), 1%–1.5% sucrose, and 0.5%–0.6% BSA.

[0139] The preparation method of the above PCR lyophilization system (reagent) is the same as in Example 3.

[0140] In the above-mentioned PCR lyophilization system (reagent), a mixture of polyethylene glycol (PEG4000), fatty alcohol polyoxyethylene ether AEO3, sucrose, and BSA serves as a protective agent during the lyophilization process. At the same time, polyethylene glycol (PEG4000), fatty alcohol polyoxyethylene ether AEO3, and sucrose ensure that the reagent forms a dense, non-porous, smooth, and neatly shaped cake-like structure during the lyophilization process, and does not deform during storage and transportation, thus ensuring the stability of its performance after reconstitution.

[0141] Example 5 A kit comprising the PCR lyophilized system (reagent) of Example 2, a reconstitution solvent (i.e., sterile DEPC double-distilled water), a positive control, and a negative control.

[0142] The positive control was prepared by diluting the positive plasmid (selected according to the target organism) with 1xTE buffer containing 0.2 w / v%–0.3 w / v% polyethylene glycol (PEG4000), 0.1 w / v%–0.15 w / v% fatty alcohol polyoxyethylene ether AEO3, and 0.2 w / v%–0.3 w / v% sucrose, so that the CT value of the liquid PCR reagent amplification was between 22 and 30, and aliquoted into 250 µL / tube. The positive control was prepared under aseptic conditions at a temperature not exceeding 4°C, and then lyophilized using the PCR lyophilization system (reagent) preparation method described in Example 3.

[0143] The negative control described above is a 1xTE buffer containing 0.2 w / v%–0.3 w / v% polyethylene glycol (PEG4000), 0.1 w / v%–0.15 w / v% fatty alcohol polyoxyethylene ether AEO3, and 0.2 w / v%–0.3 w / v% sucrose. It was dispensed in 250 µL tubes. The positive control was prepared under aseptic conditions at a temperature not exceeding 4°C, and then lyophilized using the PCR lyophilization system (reagent) preparation method described in Example 3.

[0144] Example 6 A kit comprising the PCR lyophilized system (reagent) of Example 3, a reconstitution solvent (i.e., sterile DEPC double-distilled water), a positive control (same as Example 5), and a negative control (same as Example 5).

[0145] Example 7 A kit comprising the PCR lyophilized system (reagent) of Example 4, a reconstitution solvent (i.e., sterile DEPC double-distilled water), a positive control (same as Example 5), and a negative control (same as Example 5).

[0146] Application Example 1: Application of the Full-Component Lyophilized Direct Amplification Nucleic Acid Detection Kit for Classical Swine Fever / Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (CSFV / PRRSV-M) [Reagent Specifications] 48 doses / box [Intended Use] Classical swine fever (CSF) is a disease caused by classical swine fever virus (CSFV) infection in pigs, resulting in a series of symptoms including persistent high fever, widespread systemic hemorrhage, and reproductive disorders in sows. Highly pathogenic porcine reproductive and respiratory syndrome (PRRS) is an infectious disease induced by a variant of PRRSV (PRRSV-M), characterized by reproductive disorders in sows such as fever, anorexia, abortion, stillbirth, mummified fetuses, and weak piglets, as well as respiratory symptoms and high mortality in piglets. CSFV and PRRSV-M infections in pigs exhibit similar clinical symptoms and are prone to co-infection, thus posing challenges to the diagnosis of porcine reproductive disorders. This kit is suitable for detecting CSFV and highly pathogenic PRRSV in specimens and is applicable for the auxiliary diagnosis and epidemiological investigation of CSFV and highly pathogenic PRRSV infections.

[0147]

Reagent Composition

[0148] The packaging is as shown in Table 2.

[0149] Table 2 Reagent Composition

[0150] Note: The reaction system is lyophilized powder. Add 250 µL of reconstitution solvent to the positive control and negative control lyophilized powders, mix well, and then centrifuge briefly using a handheld centrifuge before use. Components from different batches of the kit cannot be used interchangeably.

[0151]

Storage conditions and shelf life

[0152] [Applicable Instruments] ABI, Agilent MX3000P / 3005P, LightCycler, Bio-Rad, eppendorf and other series of real-time PCR instruments.

[0153] [Sample Collection] For diseased or culled pigs, collect tonsils and brain tissue, etc.; for live pigs to be tested, use a syringe to collect 5 mL of blood into a sterile centrifuge tube. Specimens can be stored at -20℃ for short periods and at -70℃ for long periods, but not for more than 6 months. Specimens should be transported using ice packs at 2-8℃, and repeated freeze-thaw cycles are strictly prohibited.

[0154]

How to Use

[0155] For both positive and negative controls, add 250 µL of reconstitution solvent, mix well, and then centrifuge briefly using a handheld centrifuge.

[0156] 2. Reagent preparation (Reagent preparation area) 2.1 Based on the total number of samples to be tested, take out the corresponding amount of CSFV / PRRSV-M reaction system. The remaining reaction system should be sealed and dried immediately. Let the required number of PCR reaction tubes be N (N = number of samples + 1 negative control tube + 1 positive control tube). The reaction system for each test is prepared as shown in Table 3.

[0157] Table 3 Reaction System

[0158] 2.2 Sample Addition (Sample Processing Area) Take 5 µL of each of the pretreated sample, positive control, and negative control, add them to the corresponding reaction tubes, cap the tubes, mix well, and centrifuge briefly.

[0159] 3. PCR amplification (nucleic acid amplification region) 3.1 Place the reaction tube to be tested into the reaction chamber of the real-time PCR instrument; 3.2 Set up the channels and sample information, and set the reaction system to 50 µL; select the fluorescence channel: For the Reporter Dye channel, select FAM (labeled CSFV probe) and VIC (labeled PRRSV probe). For the Quencher Dye channel, select NONE. Do not select ROX reference fluorescence.

[0160] 3.3 Recommended loop parameter settings are shown in Table 4.

[0161] Table 4 Loop Parameter Settings

[0162] 4. Result Analysis and Judgment 4.1 Setting Results Analysis Conditions (Please refer to the instruction manual of each instrument for settings; taking the ABI7500 instrument as an example). The results are automatically saved after the reaction is complete. Adjust the Start, End, and Threshold values ​​of the Baseline according to the analyzed image (users can adjust these values ​​as needed; the Start value can be set between 3 and 15, and the End value between 5 and 20, so that the threshold line is located in the exponential phase of the amplification curve, and the amplification curve of the control is flat or below the threshold line). Click Analyze to automatically obtain the analysis results.

[0163] 4.2 Result Interpretation: The FAM channel represents the result of classical swine fever virus detection, and the VIC channel represents the result of highly pathogenic porcine reproductive and respiratory syndrome virus detection; Positive: CT value of the detection channel ≤ 35, and the curve shows a clear exponential growth curve; Negative: CT value of the corresponding detection channel > 35 or no CT value (no amplification curve).

[0164] 4.3 Quality control standards: Negative control: no specific amplification curve or no CT value; Positive control: amplification curve with obvious exponential growth phase and CT value ≤30; All of the above conditions must be met simultaneously, otherwise the experiment is considered invalid.

[0165] Furthermore, the above samples were used as control experiments with the routine extraction and amplification stepwise liquid reagent "Chinese Classical Disease / Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (CSFV / PRRSV-M) Nucleic Acid Detection Kit (Dual Fluorescent PCR Method)" from Guangzhou Weiboxin Biotechnology Co., Ltd.

[0166] The detection results using the CSFV / PRRSV-M whole-component lyophilized direct amplification method of this invention are as follows: Figure 1 As shown, the results of the classical swine fever / highly pathogenic porcine reproductive and respiratory syndrome virus (CSFV / PRRSV-M) nucleic acid detection kit are as follows: Figure 2 As shown in the figure, the above results demonstrate superior performance compared to conventional stepwise liquid reagents for extraction and amplification.

[0167] Example 2: Application of the Canine Parvovirus / Canine Distemper Virus (CPV / CDV) Full-Component Lyophilized Direct Amplification Nucleic Acid Detection Kit [Reagent Specifications] 48 doses / box [Intended Use] Canine parvovirus disease and canine distemper virus disease are two highly contagious diseases in dogs caused by canine parvovirus (CPV) and canine distemper virus (CDV), respectively. These two diseases cause extremely high mortality rates in dogs, especially puppies, resulting in serious harm and losses. This kit is suitable for detecting canine parvovirus or canine distemper virus in diseased tissues such as the liver, spleen, and lungs, or in samples such as tears, nasal discharge, saliva, and feces. It is suitable for the auxiliary diagnosis of infections caused by these two pathogens.

[0168]

Reagent Composition

[0169] The packaging is as shown in Table 5.

[0170] Table 5 Reagent Composition

[0171] Note: The reaction system is lyophilized powder. Add 250µL of reconstitution solvent to the positive control and negative control lyophilized powders, mix well, and then centrifuge briefly using a handheld centrifuge before use. Components from different batches of the kit cannot be used interchangeably.

[0172]

Storage conditions and shelf life

[0173] [Applicable Instruments] ABI, Agilent MX3000P / 3005P, LightCycler, Bio-Rad, eppendorf and other series of real-time PCR instruments.

[0174] [Sample Collection] Liver, spleen, and lung tissues can be collected from dead dogs; blood, serum, tears, nasal discharge, saliva, and feces can be collected from live dogs. Samples can be stored at -20℃ for short periods and at -70℃ for long periods, but not exceeding 6 months. Specimens should be transported using ice packs at 2-8℃, and repeated freeze-thaw cycles are strictly prohibited.

[0175]

How to Use

[0176] 2. Reagent preparation (Reagent preparation area) 2.1 Based on the total number of samples to be tested, take out the corresponding amount of CPV / CDV reaction system. The remaining reaction system should be sealed and dried immediately. Let the required number of PCR reaction tubes be N (N = number of samples + 1 negative control tube + 1 positive control tube). The reaction system for each test is prepared as shown in Table 6.

[0177] Table 6 Reaction System

[0178] 2.2 Sample Addition (Sample Processing Area) Take 5 µL of each of the pretreated sample, positive control, and negative control, add them to the corresponding reaction tubes, cap the tubes, mix well, and centrifuge briefly.

[0179] 3. PCR amplification (nucleic acid amplification region) 3.1 Place the reaction tube to be tested into the reaction chamber of the real-time PCR instrument; 3.2 Set up the channels and sample information, and set the reaction system to 50µL; select the fluorescence channel: For the Reporter Dye channel, select FAM and VIC; for the Quencher Dye channel, select NONE. Do not select ROX reference fluorescence. 3.3 Recommended loop parameter settings are shown in Table 7.

[0180] Table 7 Recommended loop parameter settings

[0181] 4. Result Analysis and Judgment 4.1 Setting Results Analysis Conditions (Please refer to the instruction manual of each instrument for settings; taking the ABI7500 instrument as an example). The results are automatically saved after the reaction is complete. Adjust the Start, End, and Threshold values ​​of the Baseline according to the analyzed image (users can adjust these values ​​as needed; the Start value can be set between 3 and 15, and the End value between 5 and 20, so that the threshold line is located in the exponential phase of the amplification curve, and the amplification curve of the control is flat or below the threshold line). Click Analyze to automatically obtain the analysis results.

[0182] 4.2 Result Interpretation: The FAM channel represents the canine parvovirus test result, and the VIC channel represents the canine distemper virus test result; Positive: CT value of the test channel ≤ 35, and the curve shows a clear exponential growth curve; Negative: CT value of the corresponding test channel > 35 or no CT value (no amplification curve).

[0183] 4.3 Quality control standards: Negative control: no specific amplification curve or no CT value; Positive control: amplification curve with obvious exponential growth phase and CT value ≤30; All of the above conditions must be met simultaneously, otherwise the experiment is considered invalid.

[0184] Furthermore, the above samples were used as control experiments with the routine extraction and amplification stepwise liquid reagent "Canine Parvovirus / Canine Distemper Virus (CPV / CDV) Nucleic Acid Detection Kit (Dual Fluorescent PCR Method)" from Guangzhou Weiboxin Biotechnology Co., Ltd.

[0185] The detection results using the CPV / CDV whole-component lyophilized direct amplification method of this invention are as follows: Figure 3 As shown, the results of the classical swine fever / highly pathogenic porcine reproductive and respiratory syndrome virus (CPV / CDV) nucleic acid detection kit are as follows: Figure 4 As shown in the figure, the above results demonstrate superior performance compared to conventional stepwise liquid reagents for extraction and amplification.

[0186] Application Implementation Case 3: Application of the Full-Component Lyophilized Direct Amplification Nucleic Acid Detection Kit for Streptococcus suis Type 2 / Haemophilus parasuis (SS-2 / HPS) [Reagent Specifications] 48 doses / box [Intended Use] This kit is suitable for detecting Streptococcus suis type 2 and Haemophilus parasuis nucleic acid in samples. It is applicable to the auxiliary diagnosis and epidemiological investigation of Streptococcus suis type 2 and Haemophilus parasuis infection. The test results are for reference only.

[0187]

Reagent Composition

[0188] The packaging is as shown in Table 8.

[0189] Table 8 Reagent Composition

[0190] Note: The reaction system is lyophilized powder. Add 250 µL of reconstitution solvent to the positive control and negative control lyophilized powders, mix well, and then centrifuge briefly using a handheld centrifuge before use. Components from different batches of the kit cannot be used interchangeably.

[0191]

Storage conditions and shelf life

[0192] [Applicable Instruments] ABI, Agilent MX3000P / 3005P, LightCycler, Bio-Rad, eppendorf and other series of real-time PCR instruments.

[0193] [Sample Collection] For pigs that have died or been culled, collect tissue samples from the trachea, hilar lymph nodes, tonsils, lungs, heart, brain, internal organs, or muscles; for live pigs to be tested, collect pharyngeal swabs, nasal secretions, effusions, serum, or plasma. Specimens can be stored at -20℃ for short periods and at -70℃ for long periods, but not exceeding 6 months. Specimens should be transported using ice packs at 2–8℃, and repeated freeze-thaw cycles are strictly prohibited.

[0194]

How to Use

[0195] 2. Reagent preparation (Reagent preparation area) 2.1 Based on the total number of samples to be tested, take out the corresponding amount of SS-2 / HPS reaction system. The remaining reaction system should be immediately sealed and dried for storage. Let N be the number of PCR reaction tubes required (N = number of samples + 1 negative control tube + 1 positive control tube). The reaction system for each test is prepared as shown in Table 9: Table 9 Reaction System

[0196] 2.2 Sample Addition (Sample Processing Area) Take 5 µL of each of the pretreated sample, positive control, and negative control, add them to the corresponding reaction tubes, cap the tubes, mix well, and centrifuge briefly.

[0197] 3. PCR amplification (nucleic acid amplification region) 3.1 Place the reaction tube to be tested into the reaction chamber of the real-time PCR instrument; 3.2 Set up the channels and sample information, and set the reaction system to 50 µL; select the fluorescence channel: For the Reporter Dye channel, select FAM and VIC; for the Quencher Dye channel, select NONE. Do not select ROX reference fluorescence. 3.3 Recommended loop parameter settings are shown in Table 10.

[0198] Table 10 Loop Parameter Settings

[0199] 4. Result Analysis and Judgment 4.1 Setting Results Analysis Conditions (Please refer to the instruction manual of each instrument for settings; taking the ABI7500 instrument as an example). The results are automatically saved after the reaction is complete. Adjust the Start, End, and Threshold values ​​of the Baseline according to the analyzed image (users can adjust these values ​​as needed; the Start value can be set between 3 and 15, and the End value between 5 and 20, so that the threshold line is located in the exponential phase of the amplification curve, and the amplification curve of the control is flat or below the threshold line). Click Analyze to automatically obtain the analysis results.

[0200] 4.2 Result Interpretation: The FAM channel represents the detection result of Streptococcus suis type 2, and the VIC channel represents the detection result of Haemophilus parasuis; Positive: CT value of the detection channel ≤ 35, and the curve shows an obvious exponential growth curve; Negative: CT value of the corresponding detection channel > 35 or no CT value (no amplification curve).

[0201] 4.3 Quality control standards: Negative control: no specific amplification curve or no CT value; Positive control: amplification curve with obvious exponential growth phase and CT value ≤30; All of the above conditions must be met simultaneously, otherwise the experiment is considered invalid.

[0202] Furthermore, the above samples were used as control experiments with the routine extraction and amplification stepwise liquid reagent "Streptococcus suis type 2 / Haemophilus parasuis (SS-2 / HPS) Nucleic Acid Detection Kit (Dual Fluorescent PCR Method)" from Guangzhou Weiboxin Biotechnology Co., Ltd.

[0203] The detection results using the SS-2 / HPS whole-component lyophilized direct amplification method of this invention are as follows: Figure 5 As shown, the results of the classical swine fever / highly pathogenic porcine reproductive and respiratory syndrome virus (SS-2 / HPS) nucleic acid detection kit are as follows: Figure 6 As shown in the figure, the above results demonstrate superior performance compared to conventional stepwise liquid reagents for extraction and amplification.

[0204] Application Case 4: Application of the Freeze-Dried Direct Amplification Nucleic Acid Detection Kit for Shrimp White Spot Virus (WSSV) [Reagent Specifications] 48 doses / box [Intended Use] White spot disease (WSD) in shrimp is an infectious disease caused by white spot virus (WSSV). The disease is characterized by high mortality and rapid death. Common symptoms include the appearance of circular white granules or white spots on the skin of dying shrimp. Some infected shrimp may turn red or pink. Affected shrimp may exhibit lethargy, reddening or yellowing of their body color, or congregate at the edge of the pond, drastically reduce feeding, and show high mortality rates. This kit is suitable for detecting white spot virus in various shrimp tissue samples and can be used as an adjunct to the diagnosis of white spot virus infection.

[0205]

Reagent Composition

[0206] The packaging is as shown in Table 11.

[0207] Table 11 Reagent Composition

[0208] Note: The reaction system is lyophilized powder. Add 250 µL of reconstitution solvent to the positive control and negative control lyophilized powders, mix well, and then centrifuge briefly using a handheld centrifuge before use. Components from different batches of the kit cannot be used interchangeably.

[0209]

Storage conditions and shelf life

[0210] [Applicable Instruments] ABI, Agilent MX3000P / 3005P, LightCycler, Bio-Rad, eppendorf and other series of real-time PCR instruments.

[0211] [Sample Collection] For juvenile shrimp, larvae, and young shrimp, collect whole shrimp as samples. For adult shrimp, collect the epidermis, hepatopancreas, intestine, or gills as samples. Shrimp eggs can be collected directly as samples. Specimens can be stored at -20℃ for short periods and at -70℃ for long periods, but not exceeding 6 months. Specimens should be transported using ice packs at 2-8℃, and repeated freezing and thawing is strictly prohibited.

[0212]

How to Use

[0213] 2. Reagent preparation (Reagent preparation area) 2.1 Based on the total number of samples to be tested, take out the corresponding amount of WSSV reaction system. The remaining reaction system should be sealed and dried immediately. Let the required number of PCR reaction tubes be N (N = number of samples + 1 negative control tube + 1 positive control tube). The reaction system for each test is prepared as shown in Table 12 below. Table 12 Reaction System

[0214] 2.2 Sample Addition (Sample Processing Area) Take 5 µL of each of the pretreated sample, positive control, and negative control, add them to the corresponding reaction tubes, cap the tubes, mix well, and centrifuge briefly.

[0215] 3. PCR amplification (nucleic acid amplification region) 3.1 Place the reaction tube to be tested into the reaction chamber of the real-time PCR instrument; 3.2 Set up the channels and sample information, and set the reaction system to 50 µL; select the fluorescence channel: Reporter Dye: FAM; Quencher Dye: NONE; Do not select ROX reference fluorescence. 3.3 Recommended loop parameter settings are shown in Table 13.

[0216] Table 13 Cycle Parameter Settings

[0217] 4. Result Analysis and Judgment 4.1 Setting Results Analysis Conditions (Please refer to the instruction manual of each instrument for settings; taking the ABI7500 instrument as an example). The results are automatically saved after the reaction is complete. Adjust the Start, End, and Threshold values ​​of the Baseline according to the analyzed image (users can adjust these values ​​as needed; the Start value can be set between 3 and 15, and the End value between 5 and 20, so that the threshold line is located in the exponential phase of the amplification curve, and the amplification curve of the control is flat or below the threshold line). Click Analyze to automatically obtain the analysis results.

[0218] 4.2 Result Interpretation: The FAM channel represents the detection result of shrimp white spot virus; Positive: CT value of the detection channel ≤ 35, and the curve shows an obvious exponential growth curve; Negative: CT value of the corresponding detection channel > 35 or no CT value (no amplification curve).

[0219] 4.3 Quality control standards: Negative control: no specific amplification curve or no CT value; Positive control: amplification curve with obvious exponential growth phase and CT value ≤30; the above conditions should be met simultaneously, otherwise the experiment is considered invalid.

[0220] Furthermore, the above samples were used as control experiments with the conventional extraction and amplification stepwise liquid reagent "White Spot Virus (WSSV) Nucleic Acid Detection Kit (Dual Fluorescent PCR Method)" from Guangzhou Weiboxin Biotechnology Co., Ltd.

[0221] The detection results using the WSSV whole-component lyophilized direct amplification method of this invention are as follows: Figure 7 As shown, the results of the classical swine fever / highly pathogenic porcine reproductive and respiratory syndrome virus (WSSV) nucleic acid detection kit are as follows: Figure 8 As shown in the figure, the above results demonstrate superior performance compared to conventional stepwise liquid reagents for extraction and amplification.

[0222] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. An enzyme combination comprising Taq enzyme and reverse transcriptase; The amino acid sequence of the Taq enzyme is as follows: A1) SEQ ID NO:3; or A2) The amino acid sequence shown in SEQ ID NO:3 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar function; The amino acid sequence of the reverse transcriptase is as follows: B1) SEQ ID NO:6; or B2) The amino acid sequence shown in SEQ ID NO:6 is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

2. A biomaterial associated with the enzyme combination of claim 1; said biomaterial is any one of a1) to a12): a1) A nucleic acid molecule encoding the enzyme combination of claim 1; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

3. The application of the enzyme combination according to claim 1 in the preparation of the product.

4. A reagent comprising the enzyme combination and PCR amplification reaction reagent as described in claim 1.

5. The reagent according to claim 4, characterized in that, The PCR amplification reaction reagents include Tris-HCl, potassium glutamate, NH4Cl, MgCl2, and dNTPs; Preferably, the PCR amplification reaction reagents further include primers and probes for detecting the target nucleic acid.

6. The reagent according to claim 4 or 5, characterized in that, The reagents also include thiourea, dodecyl polyoxyethylene ether, sodium acetate, and urea; and / or, the reagents also include a lyophilization protectant; Preferably, the freeze-drying protectant includes polyethylene glycol, fatty alcohol polyoxyethylene ether, sucrose, and BSA.

7. A method for preparing the reagent according to any one of claims 4 to 6, comprising the following steps: Simply mix all the ingredients together; Preferably, the preparation method further includes a freeze-drying step; Preferably, the freeze-drying process includes the steps of freezing, primary drying, and desorption drying; Preferably, the freezing conditions are: maintaining 2-5°C for 8-15 minutes; and cooling to -50 to -40°C at a rate of 1-2°C / min. Preferably, the conditions for the first drying are: maintaining a temperature of -50 to -40°C for 160 to 200 min; increasing the temperature to -45 to -35°C at a rate of 0.1 to 1°C / min; and maintaining the temperature of -45 to -35°C for 700 to 750 min. Preferably, the desorption and drying conditions are as follows: heating at 0.1–1 °C / min to 20–30 °C; maintaining the temperature at 20–30 °C for 200–260 min.

8. A kit comprising the reagent, positive control, and negative control as described in any one of claims 4 to 6.

9. The use of the reagent of any one of claims 4 to 6 or the kit of claim 9 in the detection of a target, wherein the use is for purposes other than disease diagnosis.

10. A method for detecting a target analyte not for disease diagnosis, comprising the step of detecting the target analyte using the reagent of any one of claims 4 to 6 or the kit of claim 8: Preferably, the target substance or its nucleic acid is mixed with the reagents described in any one of claims 4 to 6, and then amplified by PCR.

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