PCR (Polymerase Chain Reaction) buffer solution, kit, fully premixed storage solution and application

Abstract

The invention discloses a PCR (Polymerase Chain Reaction) buffer solution, a kit, a fully premixed storage solution and application. The PCR buffer solution comprises the following components: a buffer agent, potassium salt, magnesium salt, ammonium sulfate, dNTP (T), dNTP (U), Triton X-100, dimethyl sulfoxide, tetramethylammonium chloride, glycerol, dithiothreitol and bovine serum albumin. According to the PCR buffer solution provided by the invention, the amplification efficiency of a high-GC-content template is remarkably improved through a synergistic effect generated by all the components in a specific ratio, the premixing performance is effectively enhanced, and the reagent stability after the PCR buffer solution is premixed with enzyme, primers and probes is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological products, in particular to PCR buffer, kit, fully premixed storage solution and application. BACKGROUND

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments, widely used in gene detection, gene expression analysis, genetic disease diagnosis and other fields. With the development of science and technology, PCR technology gradually develops towards multiplexing, fluorescence and high-throughput to meet the needs of rapid, real-time detection of multiple targets in complex samples. Multiplex fluorescent PCR technology can simultaneously amplify and detect multiple target fragments in a single reaction system, distinguish specific signals through different fluorescence channels, and significantly improve experimental efficiency, detection throughput and visualization of results.

[0003] However, the implementation of multiplex fluorescent PCR technology depends on the precise design and optimization of multiple primers and fluorescent probes, as well as the stability of the PCR reaction system. As an important component of the reaction system, the composition and formula of the PCR buffer directly affect the amplification efficiency, specificity and signal-to-noise ratio of the fluorescent signal.

[0004] Currently, researchers have developed a variety of optimized PCR buffers. For example:

[0005] CN111593115A provides a reaction buffer for multiplex fluorescent PCR, including the following components: HEPES 50mM, (NH4)2SO4 25mM, KCl 125mM, MgCl2 7.5mM, formamide with a volume percentage of 2.50%, dNTPs 0.3mM and dUTP 0.6mM. However, the component of this buffer system is relatively simple, it is difficult to efficiently amplify high GC content templates (such as GC content > 60%), and it cannot adapt to the direct amplification process without nucleic acid extraction, and the stability of the premixed reagent also needs to be improved.

[0006] CN119530407A discloses a composition and kit for detecting Chlamydia pneumoniae, Mycoplasma pneumoniae, and respiratory adenovirus. Specifically, the buffer components in the PCR reaction system are disclosed as follows: 50-70 mM Tris-HCl, 25-45 mM KCl, 2-2.5 mM MgCl2, 0.6-0.8 mM dNTPs, 10-20 mM (NH4)2SO4, 0.01-0.02 v% BSA, 0.02-0.05 v% Tween 20, 0.005-0.01 w% gelatin, 0.01-0.02 v% glycerol, 0.05-0.1 v% Triton-X, 0.1-0.2 v% DMSO, 8-10 mM TMAC, 1.5-3 w% trehalose, and 1-2 w% sucrose. While this buffer system can be used for sample extraction-free storage and reagent lyophilization, its amplification capacity and uniformity for templates with high GC content remain insufficient. Furthermore, its suitability and practical stability are still limited for applications where the buffer, enzyme, primers, and probes are fully premixed in liquid form and require long-term storage and stable performance.

[0007] Other literature, such as CN113195737A and CN118879681A, only involves basic buffer components and cannot improve the amplification ability for templates with high GC content. Therefore, using the above-mentioned traditional PCR buffers results in insufficient amplification ability for templates with high GC content, easily leading to uneven amplification efficiency, signal shift, or even missed detection. Furthermore, the premixing performance is poor, making it difficult to maintain the long-term stability of the reagents after premixing PCR buffers, enzymes, primers, and probes. Summary of the Invention

[0008] Therefore, it is necessary to provide PCR buffers, kits, fully premixed storage solutions, and applications.

[0009] The first aspect of this application provides a PCR buffer comprising the following components: 50 mM to 500 mM of buffer, 20 mM to 50 mM of potassium salt, 1 mM to 10 mM of magnesium salt, 5 mM to 50 mM of ammonium sulfate, 100 μM to 500 μM of dNTP(T), 100 μM to 500 μM of dNTP(U), 0.05% (v / v) to 0.2% (v / v) of Triton X-100, 1% (v / v) to 5% (v / v) of dimethyl sulfoxide, 20 mM to 70 mM of tetramethylammonium chloride, 1% (v / v) to 5% (v / v) of glycerol, 1 mM to 5 mM of dithiothreitol, and 0.1 mg / mL to 0.5 mg / mL of bovine serum albumin.

[0010] In some implementations, one or more of the following features are satisfied:

[0011] The buffer includes HEPES buffer;

[0012] The pH of the PCR buffer is 7.5-9.0;

[0013] The potassium salt is potassium chloride, and the magnesium salt is magnesium chloride.

[0014] A second aspect of this application provides a fully premixed PCR storage solution comprising the PCR buffer described in the first aspect of this application, wherein the fully premixed PCR storage solution further comprises PCR amplification reagents; optionally, the PCR amplification reagents comprise at least one of DNA polymerase, reverse transcriptase, UDG enzyme, primers, and probes.

[0015] A third aspect of this application provides a PCR detection kit comprising at least one of the PCR buffer described in the first aspect of this application and the PCR fully premixed storage solution described in the second aspect of this application.

[0016] In some embodiments, the PCR test kit further includes a nucleic acid release agent.

[0017] The fourth aspect of this application provides the application of the PCR fully premixed storage solution described in the second aspect of this application or the PCR detection kit described in the third aspect of this application in amplifying nucleic acid samples. The application is for non-diagnostic purposes and includes: mixing the premixed and stored PCR fully premixed storage solution with a nucleic acid sample to obtain a PCR mixed sample, and performing PCR amplification; wherein the nucleic acid sample includes at least two nucleic acids, and the PCR fully premixed storage solution is stored for not less than 7 days.

[0018] In some embodiments, the nucleic acid sample includes at least one of DNA and RNA samples; alternatively, the nucleic acid sample includes at least two different pathogens.

[0019] In some embodiments, the fully premixed PCR storage solution is stored at 4°C for at least 7 days, or at -20°C for at least 30 days.

[0020] In some embodiments, the PCR fully premixed storage solution is further subjected to a nucleic acid release process before being mixed with the nucleic acid sample.

[0021] The fifth aspect of this application provides an application for direct amplification of nucleic acid samples based on PCR buffer for non-diagnostic purposes, including:

[0022] The nucleic acid sample to be tested is mixed with a nucleic acid releasing agent to obtain a nucleic acid solution;

[0023] The nucleic acid solution was mixed with the PCR fully premixed storage solution described in the second aspect of this application and then subjected to direct PCR amplification.

[0024] Optionally, the nucleic acid sample to be tested includes at least two different pathogens, including at least two of adenovirus, human rhinovirus, and Mycoplasma pneumoniae.

[0025] The aforementioned PCR buffer significantly improves the amplification efficiency of templates with high GC content (e.g., GC content > 60%) through the synergistic effect of each component in a specific ratio, and effectively enhances the premixing performance, significantly improving the reagent stability after the PCR buffer is premixed with enzymes, primers and probes. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0027] Figure 1 The amplification results of PCR buffer groups #1-5 in one embodiment of this application;

[0028] Figure 2 The amplification results of PCR buffer groups #1, #6 and #7 in one embodiment of this application;

[0029] Figure 3 This is the amplification result of PCR buffer group #1 before and after premixing at 4°C for 7 days (d) in one embodiment of this application;

[0030] Figure 4 This is the amplification result of PCR buffer group #8 before and after premixing at 4°C for 7 days in one embodiment of this application;

[0031] Figure 5 This is the amplification result of PCR buffer group #9 before and after premixing at 4°C for 7 days in one embodiment of this application;

[0032] Figure 6 This is the amplification result of PCR buffer group #10 before and after premixing at 4°C for 7 days in one embodiment of this application;

[0033] Figure 7 This is the amplification result of PCR buffer group #11 before and after premixing at 4°C for 7 days in one embodiment of this application;

[0034] Figure 8 This is the amplification result of PCR buffer group #12 before and after premixing at 4°C for 7 days in one embodiment of this application;

[0035] Figure 9 This is the amplification result of PCR buffer group #1 before and after premixing at -20℃ for 30 days in one embodiment of this application;

[0036] Figure 10 This is the amplification result of PCR buffer group #8 before and after premixing at -20℃ for 30 days in one embodiment of this application;

[0037] Figure 11 This is the amplification result of PCR buffer group #9 before and after premixing at -20℃ for 30 days in one embodiment of this application;

[0038] Figure 12 This is the amplification result of PCR buffer group #10 in one embodiment of this application before and after premixing at -20℃ for 30 days;

[0039] Figure 13 This is the amplification result of PCR buffer group #11 in one embodiment of this application before and after premixing at -20℃ for 30 days;

[0040] Figure 14 This is the amplification result of PCR buffer group #12 in one embodiment of this application before and after premixing at -20℃ for 30 days;

[0041] Figure 15 The results of amplification of rhinovirus products with different GC contents using PCR buffers in groups #12, #13, and #14 of one embodiment of this application are shown. Detailed Implementation

[0042] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0044] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0045] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0046] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0047] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0048] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0049] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0050] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0051] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0052] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0053] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C.

[0054] In this application, w / v represents mass-volume percentage. In some embodiments of this application, w is expressed in grams and v in mL.

[0055] In this application, the concentration or amount of each component in the buffer solution, unless otherwise defined, refers to the final concentration or amount in the buffer solution, which is equivalent to the final concentration or amount in the reagents constituting the buffer solution.

[0056] Currently, traditional multiplex PCR buffers are used, but their amplification capacity for templates with high GC content is insufficient, which can easily lead to uneven amplification efficiency, signal shift, or even missed detection. Furthermore, their premixing performance is poor, making it difficult to maintain the long-term stability of the reagents after premixing PCR buffers, enzymes, primers, and probes.

[0057] Based on this, the embodiments of this application at least provide PCR buffer, kit, fully premixed storage solution and application.

[0058] In this application, "direct amplification" refers to the nucleic acid release and direct amplification technology that does not require sample extraction. It means that the nucleic acid of the sample is directly amplified and detected without the need for nucleic acid extraction or purification.

[0059] In this application, "fully premixed" refers to a reagent prepared by pre-mixing all active ingredients and reaction components (except template nucleic acid) required to complete a PCR reaction. When using it, you only need to mix the fully premixed solution with the processed sample (i.e., template nucleic acid) and you can directly perform PCR amplification without any additional preparation, dilution or mixing steps.

[0060] In this application, "multiplex fluorescent PCR" is a technique for simultaneously detecting multiple target nucleic acid sequences in a single reaction system. It achieves simultaneous amplification and qualitative / quantitative analysis of multiple targets by adding specific primers for different targets and probes with different fluorescent labels to the reaction.

[0061] In this application, "dNTP(T)" refers to a mixture of dTTP, dATP, dCTP, and dGTP; "dNTP(U)" refers to a mixture of dUTP, dATP, dCTP, and dGTP.

[0062] In a first aspect of this application, a PCR buffer composition is provided, comprising a buffer, a surfactant, a potassium salt, a magnesium salt, dNTP(T), dNTP(U), ammonium sulfate, a protectant, and an enhancer, wherein the surfactant comprises Triton X-100, the protectant comprises one or more of glycerol, bovine serum albumin, and dithiothreitol, and the enhancer comprises one or more of dimethyl sulfoxide, betaine, and tetramethylammonium chloride, wherein the mass ratio of tetramethylammonium chloride to dithiothreitol is (2~100):1.

[0063] In some embodiments, the mass ratio of tetramethylammonium chloride to dithiothreitol can be, but is not limited to, 2:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or any ratio or range between two of the above.

[0064] In a second aspect of this application, a PCR buffer is provided, comprising the above-described PCR buffer composition and an aqueous solvent; further, the aqueous solvent comprises nuclease-free water.

[0065] In some implementations, the PCR buffer provided in this application is suitable for multiplex fluorescent PCR.

[0066] It should be noted that the PCR buffer provided in this application significantly improves the amplification efficiency of templates with high GC content (such as GC content > 60%) through the synergistic effect of each component under specific ratios, and effectively enhances the premixing performance. It also significantly improves the reagent stability after the PCR buffer is premixed with enzymes, primers and probes. For example, the fully premixed solution can be stored at 4°C for at least 7 days and at -20°C for at least 30 days without affecting the detection performance.

[0067] In some embodiments, the PCR buffer comprises the following: buffer, potassium salt, magnesium salt, ammonium sulfate ((NH4)2SO4), dNTP(T), dNTP(U), Triton X-100, DMSO (dimethyl sulfoxide), glycerol, tetramethylammonium chloride (TMAC), DTT (dithiothreitol), and BSA (bovine serum albumin), and also includes nuclease-free water. The buffer can be Tris-HCl buffer, Tris-acetic acid buffer, HEPES buffer, Tricine buffer, or Bicine buffer. In some embodiments, the buffer is HEPES buffer.

[0068] In some embodiments, the PCR buffer is composed of the following: buffer, potassium salt, magnesium salt, ammonium sulfate ((NH4)2SO4), dNTP(T), dNTP(U), Triton X-100, DMSO (dimethyl sulfoxide), glycerol, tetramethylammonium chloride (TMAC), DTT (dithiothreitol), and BSA (bovine serum albumin), with the remaining component being nuclease-free water.

[0069] In some embodiments, the PCR buffer composition is as follows: containing 50 mM–500 mM Tris-HCl buffer, or 50 mM–500 mM Tris-acetic acid buffer, or 50 mM–500 mM HEPES buffer, or 50 mM–500 mM Tricine buffer, or 50 mM–500 mM Bicine buffer, 20 mM–50 mM potassium salt, 1 mM–10 mM magnesium salt, 5 mM–50 mM ammonium sulfate ((NH4)2SO4), 100 μM–500 μM dNTP(T), 100 μM–500 μM dNTP(U), and 0.05%–0.2% vol Triton. X-100, 1%~5% vol DMSO (dimethyl sulfoxide), 1%~5% vol glycerol, 20 mM~70 mM tetramethylammonium chloride (TMAC), 1~5 mM DTT (dithiothreitol), and 0.1 mg / mL~0.5 mg / mL BSA (bovine serum albumin), and also includes nuclease-free water; in some embodiments, the remaining components are nuclease-free water.

[0070] Non-limiting examples of potassium salts may include potassium chloride.

[0071] Non-limiting examples of magnesium salts may include magnesium chloride.

[0072] In some embodiments, the PCR buffer composition is as follows: containing 50 mM–500 mM Tris-HCl buffer, or 50 mM–500 mM Tris-acetic acid buffer, or 50 mM–500 mM HEPES buffer, or 50 mM–500 mM Tricine buffer, or 50 mM–500 mM Bicine buffer, 20 mM–50 mM KCl, 1 mM–10 mM MgCl2, 5 mM–50 mM ammonium sulfate ((NH4)2SO4), 100 μM–500 μM dNTP(T), 100 μM–500 μM dNTP(U), and 0.05%–0.2% vol Triton. X-100, 1%~5% vol DMSO (dimethyl sulfoxide), 1%~5% vol glycerol, 20 mM~70 mM tetramethylammonium chloride (TMAC), 1~5 mM DTT (dithiothreitol), and 0.1 mg / mL~0.5 mg / mL BSA (bovine serum albumin), with the remainder being nuclease-free water.

[0073] In some embodiments, a multiplex fluorescent PCR buffer is provided, comprising the following components: 50 mM to 500 mM of buffer, 20 mM to 50 mM of potassium salt, 1 mM to 10 mM of magnesium salt, 5 mM to 50 mM of ammonium sulfate, 100 μM to 500 μM of dNTP(T), 100 μM to 500 μM of dNTP(U), 0.05% (v / v) to 0.2% (v / v) of Triton X-100, 1% (v / v) to 5% (v / v) of dimethyl sulfoxide, 20 mM to 70 mM of tetramethylammonium chloride, 1% (v / v) to 5% (v / v) of glycerol, 1 mM to 5 mM of dithiothreitol, and 0.1 mg / mL to 0.5 mg / mL of bovine serum albumin.

[0074] In some implementations, the concentration of HEPES buffer in the multiplex fluorescent PCR buffer can be, but is not limited to, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or any value or range between two of the above.

[0075] In some implementations, the concentration of potassium salt (such as KCl) in the multiplex fluorescent PCR buffer can be, but is not limited to, 20 mM, 30 mM, 40 mM, 50 mM, or any value or range between two of the above.

[0076] In some implementations, the concentration of magnesium salt (such as MgCl2) in the multiplex fluorescent PCR buffer can be, but is not limited to, 1 mM, 5 mM, 10 mM, or any value or range between two of the above.

[0077] In some embodiments, the concentration of ammonium sulfate in the multiplex fluorescent PCR buffer can be, but is not limited to, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, or any value or range between two of the above.

[0078] In some implementations, the concentration of dNTP(T) in the multiplex fluorescent PCR buffer can be, but is not limited to, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, or any value or range between two of the above.

[0079] In some implementations, the concentration of dNTP(U) in the multiplex fluorescent PCR buffer can be, but is not limited to, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, or any value or range between two of the above.

[0080] In some implementations, the concentration of Triton X-100 in the multiplex fluorescent PCR buffer can be, but is not limited to, 0.05% (v / v), 0.1% (v / v), 0.15% (v / v), 0.2% (v / v), or any value or range between two of the above.

[0081] In some implementations, the concentration of dimethyl sulfoxide in the multiplex fluorescent PCR buffer can be, but is not limited to, 1% (v / v), 2% (v / v), 3% (v / v), 4% (v / v), 5% (v / v), or any value or range between two of the above.

[0082] In some implementations, the concentration of glycerol in the multiplex fluorescent PCR buffer can be, but is not limited to, 1% (v / v), 2% (v / v), 3% (v / v), 4% (v / v), 5% (v / v), or any value or range between two of the above.

[0083] In some implementations, the concentration of tetramethylammonium chloride in the multiplex fluorescent PCR buffer can be, but is not limited to, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or any value or range between two of the above.

[0084] In some embodiments, the concentration of BSA in the multiplex fluorescent PCR buffer can be, but is not limited to, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, or any value or range between two of the above.

[0085] In some embodiments, one of the PCR buffer components and their proportions are: 100 mM HEPES buffer, 50 mM KCl, 6 mM MgCl2, 5 mM ammonium sulfate, 100 μM dNTP(T), 200 μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, 0.2 mg / mL BSA, 5% vol glycerol, 50 mM TMAC, and 1 mM DTT, with the remainder being nuclease-free water.

[0086] In some embodiments, the pH of the PCR buffer is controlled within the range of 7.5-9.0. The preparation steps are as follows: 1. Weigh or dilute HEPES, KCl, MgCl2, ammonium sulfate, dNTP(T), dNTP(U), Triton X-100, DMSO, glycerol, TMAC, DTT, and BSA according to the above concentrations, and dissolve them in nuclease-free water; 2. Adjust the pH of the dissolved solution to 7.5-9.0 (it can be adjusted to around 8.5) using a pH meter; 3. Sterilize the pH-adjusted solution by passing it through a 0.22 μM aqueous filter membrane to obtain the PCR buffer.

[0087] It should be noted that the HEPES buffer in the above PCR buffer maintains pH stability over a wide temperature range; it can maintain a suitable pH for polymerase activity during PCR heating and cooling.

[0088] In a third aspect of this application, a multiplex fluorescent PCR premix is ​​provided, comprising the aforementioned multiplex PCR buffer and PCR amplification reagents; further, the PCR amplification reagents comprise at least one of DNA polymerase, reverse transcriptase, UDG enzyme, primers, and probes.

[0089] It should be noted that the multiplex fluorescent PCR premix provided in this application mixes the PCR reaction solution, enzymes and fluorescent probes in advance. When using it, you only need to add the sample to amplify and detect it. It can maintain stable detection performance for a long time after storage, which meets the high requirements of clinical testing for ease of operation and consistency of results.

[0090] In a fourth aspect of this application, a multiplex fluorescent PCR detection kit is provided, comprising at least one of the above-described multiplex fluorescent PCR buffer composition, the above-described multiplex fluorescent PCR buffer, and the above-described multiplex fluorescent PCR premix.

[0091] In some implementations, the multiplex fluorescent PCR detection kit also includes a nucleic acid release agent.

[0092] In a fifth aspect of this application, the application of the above-mentioned multiplex fluorescent PCR premix or the above-mentioned multiplex fluorescent PCR detection kit in amplifying nucleic acid samples is provided, comprising: mixing the premixed and stored multiplex fluorescent PCR premix with a nucleic acid sample to obtain a PCR mixed sample, and performing PCR amplification; wherein the nucleic acid sample includes at least two nucleic acids, and the PCR premix is ​​stored for not less than 7 days.

[0093] It should be noted that "at least 2 nucleic acids" means at least 2 different target genes or nucleic acid sequences.

[0094] In some implementations, the nucleic acid sample includes at least one of DNA and RNA samples; further, the nucleic acid sample includes at least two different pathogens.

[0095] In some implementations, the PCR premix is ​​stored at 4°C for at least 7 days, or at -20°C for at least 30 days.

[0096] In some implementations, the above applications are not for diagnostic purposes, but can be used for public health and food safety monitoring, as well as animal and plant quarantine and healthy breeding.

[0097] In some implementations, the above application is for diagnostic purposes.

[0098] In a sixth aspect of this application, a method for direct amplification of nucleic acid samples using multiplex fluorescent PCR buffer is provided, comprising:

[0099] S100: Mix the nucleic acid sample to be tested with the nucleic acid release agent to obtain a nucleic acid solution;

[0100] S200: Mix the nucleic acid solution with the above-mentioned multiplex fluorescent PCR premix and then perform direct PCR amplification.

[0101] In some implementations, the nucleic acid sample to be tested is a sputum sample.

[0102] In some implementations, the nucleic acid sample to be tested includes at least two different pathogens.

[0103] In some implementations, the pathogen includes at least two of adenovirus, human rhinovirus, and Mycoplasma pneumoniae.

[0104] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0105] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0106] The [multiplex fluorescence PCR buffer] used in the following examples is as follows: containing 50mM~500mM Tris-HCl buffer, or 50mM~500mM Tris-acetic acid buffer, or 50mM~500mM HEPES buffer, or 50mM~500mM Tricine buffer, or 50mM~500mM Bicine buffer, 20mM~50mM KCl, 1mM~10mM MgCl2, 5mM~50mM ammonium sulfate ((NH4)2SO4), 100μM~500μM dNTP(T), 100μM~500μM dNTP(U), and 0.05%~0.2% vol Triton. X-100, 1%~5% vol DMSO (dimethyl sulfoxide), 1~3M betaine, 1%~5% vol glycerol, 20mM~70mM tetramethylammonium chloride (TMAC), 1~5mM DTT (dithiothreitol), and 0.1mg / mL~0.5mg / mL BSA (bovine serum albumin), with the remainder being nuclease-free water.

[0107] Detection method: Multiplex fluorescent PCR detection.

[0108] In detail, specific primer pairs and probes are designed for each target gene to be detected. Different probes are labeled with non-interfering fluorescent reporter groups (such as FAM, VIC, ROX, CY5, etc.). During the PCR reaction, each probe binds only to its corresponding target sequence. When the probe is digested by Taq enzyme, it releases specific fluorescence. The instrument can distinguish different wavelengths of fluorescence signals and simultaneously record the amplification curves of each target, calculating their respective Ct values. The Ct value represents the number of cycles required for the fluorescence signal in each reaction tube to reach a set threshold. The Ct value of each template has a linear relationship with the logarithm of the initial copy number of that template. The Ct value reflects the concentration of the sample being detected; a lower Ct value indicates a higher nucleic acid concentration, and a higher Ct value indicates a lower nucleic acid concentration.

[0109] The core advantage of this technology lies in its high efficiency—multiple targets can be detected in a single reaction, significantly saving sample volume, reagents, and time; while maintaining the high specificity and accuracy of singleton qPCR. The key is that primer and probe design must avoid cross-interference (such as primer dimers and non-specific probe binding), and the PCR reaction system (such as buffer components and Mg²⁺ concentration) must be optimized to balance the amplification efficiency of multiple targets.

[0110] Unless otherwise specified, all test materials used in the following examples are commercially available products.

[0111] Example 1

[0112] A quadruple fluorescent PCR method was used to test the direct amplification efficiency of the PCR buffer. The quadruple fluorescent PCR included detection of adenovirus (ADV), human rhinovirus (HRV), Mycoplasma pneumoniae (MP), and endogenous GAPDH as an internal standard (IC). Specifically, the fluorescent reporter group for the adenovirus probe was FAM, and the quencher group was BHQ1; the fluorescent reporter group for the human rhinovirus probe was HEX, and the quencher group was BHQ1; the fluorescent reporter group for the GAPDH gene was ROX, and the quencher group was BHQ2; and the fluorescent reporter group for Mycoplasma pneumoniae was CY5, and the quencher group was BHQ2. The same primer-probe mixture was used to prepare PCR mixtures #1-#5, along with the same set of enzyme mixtures (including Taq polymerase, RT-PCR, and UDG enzyme), which were named PCR mixture 1-PCR mixture 5.

[0113] ADV, HRV, and MP positive samples were mixed to prepare a pooled sample. Nucleic acid release was performed using the nucleic acid release agent (direct amplification, extraction-free) from Sansure Biotech Inc., following its instructions. The pooled sample was then subjected to PCR amplification using PCR mixture 1 through PCR mixture 5, with the results repeated three times.

[0114] The formulations for PCR buffers (buffer system tests) for groups #1-5 are as follows:

[0115] #1 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO and 50mM TMAC, the remaining components are nuclease-free water, and the pH of the buffer is adjusted to 8.5 with 1M HCl and 1M NaOH.

[0116] #2 PCR buffer: 100mM Tricine buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0117] #3 PCR buffer: 100mM Bicine buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0118] #4 PCR buffer: 100mM Tris-HCl buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0119] #5 PCR buffer: 100mM Tris-acetic acid buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M acetic acid and 1M NaOH.

[0120] The amplification results of PCR buffers #1-5 are as follows: Figure 1 As shown in Table 1 (one curve is selected for each target point in each group), the Ct values ​​(average value) are shown in Table 1.

[0121] Table 1

[0122]

[0123] The results showed that the direct amplification effect of PCR buffer group #1 was better than that of the other four buffer groups at all target sites.

[0124] To investigate the optimal pH value of PCR buffer group #1, PCR buffer groups #6 and #7 were also prepared. The formulations of PCR buffer groups #6 and #7 are as follows:

[0125] #6 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO and 50mM TMAC, the remaining components are nuclease-free water, and the pH of the buffer is adjusted to 8.0 with 1M HCl and 1M NaOH.

[0126] #7 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 9.0 with 1M HCl and 1M NaOH.

[0127] Mix PCR buffers #1, #6, and #7 with the same primer and probe mixture, and then with the same enzyme mixture (including Taq enzyme, RT enzyme, and UDG enzyme) to prepare PCR mixtures, which are named PCR mixture 1, PCR mixture 6, and PCR mixture 7, respectively.

[0128] ADV, HRV, and MP positive samples were mixed to prepare a pooled sample. Nucleic acid release was performed using the nucleic acid release agent (direct amplification, extraction-free) from Sansure Biotech Inc., following its instructions. The pooled sample was then mixed with PCR mixture 1 (before and after premixing), PCR mixture 6, and PCR mixture 7 for PCR amplification, with the results repeated three times.

[0129] The amplification results of PCR buffers in groups #1, #6, and #7 are as follows: Figure 2 As shown in Table 2 (one curve is selected for each target point in each group), the Ct values ​​(average value) are shown in Table 2.

[0130] Table 2

[0131]

[0132] The results showed that the direct amplification effect of PCR buffer group #1 was superior to that of buffer groups #6 and #7 at all target sites. Therefore, PCR buffer group #1 was selected for subsequent premixed tests, and some additives were added to PCR buffer group #1 for further testing, which was named PCR buffer groups #8-12.

[0133] The PCR buffer formulations for groups #8-12 are as follows:

[0134] #8 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 1M betaine, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0135] #9 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO, 1M betaine, and 50mM TMAC. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0136] #10 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, 50mM TMAC, and 5% vol glycerol. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0137] #11 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1% vol Triton X-100, 2% vol DMSO, 50mM TMAC, 5% vol glycerol, and 1mM DTT. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0138] #12 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO, 50mM TMAC, 5%vol glycerol, 1mM DTT, and 0.2mg / mL BSA. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0139] PCR buffers #1 and #8-12 were mixed with the same primer-probe mixture, and then combined with the same enzyme mixture (including Taq enzyme, RT enzyme, and UDG enzyme) to prepare PCR mixtures, which were named PCR Mixture 1, PCR Mixture 8-PCR Mixture 12, respectively. The PCR reaction solutions were premixed at 4℃ and -20℃, respectively, for 7 days at 4℃ and 30 days at -20℃.

[0140] After premixing, ADV, HRV, and MP positive samples were mixed to prepare a pooled sample. Nucleic acid release was performed using the nucleic acid release agent (direct amplification, extraction-free) from Sansure Biotech Inc., following its instructions. The pooled sample was then mixed with PCR mixture 1 (before and after premixing) and PCR mixture 6-10 for PCR amplification, with the results repeated three times.

[0141] The amplification results of PCR buffers in groups #1 and #8-12 before and after 7 days of premixing at 4℃ are as follows: Figures 3-8 As shown in Table 3 (one curve is selected for each target point in each group), the Ct values ​​(average value) are shown in Table 3.

[0142] Table 3

[0143]

[0144] The results showed that when DMSO was replaced with betaine, which has a similar function, or when both DMSO and betaine were present in group #9, the effect of buffer #8 was not significantly improved compared to buffer #1 or was slightly worse. There was no significant difference in the Ct value and fluorescence intensity of the four target sites before and after premixing the PCR buffer at 4℃ for 7 days. Moreover, whether freshly prepared or premixed at 4℃ for 7 days, the effect of PCR buffer #12 was better than other groups, mainly in that the Ct value was earlier and the amplification curve was more prominent.

[0145] The amplification results of PCR buffers in groups #1 and #8-12 before and after 30 days of premixing at -20℃ are as follows: Figures 9-14 As shown in Table 4 (one curve is selected for each target point in each group), the Ct values ​​(average value) are shown in Table 4.

[0146] Table 4

[0147]

[0148] The results showed that, similar to the results of premixing at 4 degrees Celsius, the performance of buffer #8 was not significantly improved or was slightly worse than that of buffer #1 when DMSO was replaced with betaine, which has a similar function, or when both DMSO and betaine were present in group #9. The Ct values ​​and fluorescence intensities of the four target sites of PCR buffer #12 were not significantly different before and after premixing at -20℃ for 30 days. Moreover, the performance of buffer #12 was better than that of other groups, whether it was freshly prepared or after premixing at -20℃ for 30 days, mainly in that the Ct values ​​were higher and the amplification curves were more prominent.

[0149] Considering that many primer and probe designs in clinical settings may have excessively high GC content (>60%), and that some pathogens have numerous mutations or subtypes, requiring primers to incorporate many degenerate bases, the optimal concentration of TMAC was optimized to ensure the system can accommodate these complex and diverse templates without inhibiting the PCR reaction. Two sets of systems were tested. The difference between PCR buffer sets #13 and #14 and PCR buffer set #12 lies in the TMAC concentration; all other components and pH values ​​are identical, as detailed below:

[0150] #13 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO, 10mM TMAC, 5%vol glycerol, 1mM DTT, and 0.2mg / mL BSA. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0151] #14 PCR buffer: 100mM HEPES buffer, 50mM KCl, 6mM MgCl2, 5mM ammonium sulfate, 100μM dNTP(T), 200μM dNTP(U), 0.1%vol Triton X-100, 2%vol DMSO, 80mM TMAC, 5%vol glycerol, 1mM DTT, and 0.2mg / mL BSA. The remaining components are nuclease-free water. The pH of the buffer was adjusted to 8.5 with 1M HCl and 1M NaOH.

[0152] Two new sets of rhinovirus primers and probes were designed. The first set of primers and probes had a GC content of 45% and no degenerate bases, while the second set of rhinovirus primers and probes had a GC content >60% and three degenerate bases on each of the upstream and downstream primers. The fluorescent reporter group for both sets was CY5, and the quencher group was BHQ2. PCR buffers (sets #12, #13, and #14) were mixed with the primer and probe mixtures from both sets, and then combined with the same enzyme mixture (including Taq polymerase, RT enzyme, and UDG enzyme) to prepare PCR mixtures.

[0153] HRV-positive samples were subjected to nucleic acid release using the nucleic acid release agent (direct amplification, extraction-free) from Sansure Biotech Inc., following the manufacturer's instructions. The agent was then diluted to a low-concentration template (Ct value > 30). Samples were amplified using the three PCR mixtures described above, with each mixture being applied in 20 replicates.

[0154] The amplification results of PCR buffers in groups #12, #13, and #14 are as follows: Figure 15 As shown in Table 5, the Ct values ​​are as follows.

[0155] Table 5

[0156]

[0157] The results showed that #12 buffer performed well in amplifying both products with normal and high GC content; while #13 buffer performed well in detecting products with normal GC content, but Ct was delayed and there were missed detections when detecting high GC content templates; and #13 buffer, due to its high concentration, performed poorly in detecting both products with normal and high GC content.

[0158] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0159] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A PCR buffer, characterized in that, The PCR buffer contains the following components: 50mM~500mM buffer, 20mM~50mM potassium salt, 1mM~10mM magnesium salt, 5mM~50mM ammonium sulfate, 100μM~500μM dNTP(T), 100μM~500μM dNTP(U), 0.05% (v / v)~0.2% (v / v) Triton X-100, 1% (v / v)~5% (v / v) dimethyl sulfoxide, 20mM~70mM tetramethylammonium chloride, 1% (v / v)~5% (v / v) glycerol, 1mM~5mM dithiothreitol, and 0.1mg / mL~0.5mg / mL bovine serum albumin.

2. The PCR buffer as described in claim 1, characterized in that, It meets one or more of the following characteristics: The buffer includes HEPES buffer; The pH of the PCR buffer is 7.5-9.0; The potassium salt is potassium chloride; The magnesium salt is magnesium chloride.

3. A fully premixed PCR storage solution, characterized in that, It includes the PCR buffer according to any one of claims 1 to 2, and the PCR fully premixed storage solution further includes PCR amplification reagents.

4. The PCR fully premixed storage solution as described in claim 3, characterized in that, The PCR amplification reagents include at least one of DNA polymerase, reverse transcriptase, UDG enzyme, primers, and probes.

5. A PCR detection kit, characterized in that, It includes at least one of the PCR buffer according to any one of claims 1 to 2 and the PCR fully premixed storage solution according to any one of claims 3 to 4.

6. The application of the PCR fully premixed storage solution according to any one of claims 3-4 or the PCR detection kit according to claim 5 in amplifying nucleic acid samples, characterized in that, The application is for non-diagnostic purposes and includes: The PCR fully premixed storage solution, after premixing and storage, is mixed with a nucleic acid sample to obtain a PCR mixed sample, which is then subjected to PCR amplification; wherein the nucleic acid sample includes at least two types of nucleic acids, and the PCR fully premixed storage solution is stored for no less than 7 days.

7. The application as described in claim 6, characterized in that, The nucleic acid sample includes at least one of DNA and RNA samples, and / or the nucleic acid sample includes at least two different pathogens.

8. The application as described in claim 6 or 7, characterized in that, The fully premixed PCR storage solution is stored at 4°C for no less than 7 days, or at -20°C for no less than 30 days.

9. An application of direct amplification of nucleic acid samples based on PCR buffer for non-diagnostic purposes, characterized in that, include: The nucleic acid sample to be tested is mixed with a nucleic acid releasing agent to obtain a nucleic acid solution; The nucleic acid solution is mixed with the PCR fully premixed storage solution according to any one of claims 3 to 4 and then subjected to direct PCR amplification.

10. The application of non-diagnostic PCR buffer-based direct amplification nucleic acid samples as described in claim 9, characterized in that, The nucleic acid sample to be tested includes at least two different pathogens, including at least two of adenovirus, human rhinovirus, and Mycoplasma pneumoniae.

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