Pcr amplification composition and its use in rapid pcr amplification detection

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a PCR amplification composition and application thereof in rapid PCR amplification detection. The application provides a PCR amplification composition, which comprises PEG10000 or PEG20000 and a PCR amplification reaction solution used in cooperation with the PEG10000 or PEG20000. The PCR amplification composition uses high-molecular-weight PEG as a "molecular crowding agent", increases the molecular crowding degree of a reaction system, promotes the combination of a DNA polymerase and a template, improves the catalytic efficiency of the enzyme, and thus completes a high-efficiency amplification reaction in a shorter denaturation and annealing time. In addition, the high-molecular-weight PEG can form a stable enzyme-template complex, reduces the denaturation and inactivation of the enzyme in the rapid temperature rising and falling process, and guarantees the specificity and yield of amplification.

Description

Technical Field

[0001] This application belongs to the field of molecular biology technology, specifically relating to a PCR amplification composition and its application in rapid PCR amplification detection. Background Technology

[0002] Since its invention in 1985, polymerase chain reaction (PCR) has become a core tool in modern molecular diagnostics, genetic engineering, and life science research. Its name comes from one of its key components—DNA polymerase—which amplifies specific DNA fragments through in vitro enzymatic replication. As PCR proceeds, the newly generated DNA (i.e., amplicon) serves as a template for the next round of replication, thus initiating a chain reaction that causes the DNA template to amplify exponentially. Using PCR, single or extremely small copies of DNA can be amplified millions of times or even more. A PCR system typically includes a thermostable DNA polymerase, dNTPs, and a pair of primers. For RNA samples, an RT enzyme is added to the PCR system to reverse transcribe the RNA into cDNA, which is then used for PCR detection.

[0003] Under the combined action of thermostable DNA polymerase, dNTPs, and a pair of primers, the target fragment undergoes exponential amplification through a three-step cycle of denaturation, annealing, and extension. This "equilibrium paradigm" is easy to understand but does not conform to physical reality: temperature cannot be switched instantaneously, and the reaction rates vary with temperature. Once the primers anneal, polymerase extension begins immediately. Therefore, a more accurate description for rapid PCR is the "kinetic paradigm": the reaction rate and temperature are always in dynamic change, without the need for a fixed temperature, as long as the product denatures and the primers anneal. However, as applications shift from the laboratory to point-of-care testing, on-site quarantine, and immediate surgical decision-making, the traditional "slow PCR" of 1-2 hours and 30-40 cycles can no longer meet the time-sensitive requirements.

[0004] It is evident that traditional rapid PCR technology suffers from problems such as long amplification time, typically requiring 1-2 hours to complete 30-40 cycles, which cannot meet the needs of applications with high timeliness requirements and low amplification efficiency. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a PCR amplification composition and its application in rapid PCR amplification detection.

[0006] In another aspect, this application provides a PCR amplification composition, including a PCR amplification reaction solution and PEG10000 or PEG20000;

[0007] The PCR amplification reaction solution includes one or more of the following: 10 mM - 30 mM Tris-Cl, pH 8.5; 30 mM - 100 mM KCl; 3 mM - 10 mM MgCl2; 0.5 mM - 2 mM DTT; 0.05 v / v% - 0.5 v / v% Tween-20; 0.3 mM - 0.6 mM dNTPmix; and 0.3 mM - 0.5 mM dUTP.

[0008] In some embodiments, the final concentration of PEG20000 in the PCR amplification composition is 2.2 w / v%-2.6 w / v.

[0009] In some embodiments, the final concentration of PEG10000 in the PCR amplification composition is 2.2 w / v%-2.6 w / v.

[0010] In some embodiments, the PCR amplification reaction solution also includes bovine serum albumin.

[0011] In some embodiments, the final concentration of bovine serum albumin in the PCR amplification composition is 0.5 mg / mL to 2 mg / mL.

[0012] In some embodiments, the PCR amplification composition further includes an enzyme composition.

[0013] In some embodiments, the enzyme composition comprises one or more of UDG at 0.5 U / test-2 U / test, Taq enzyme at 3 U / test-10 U / test, RT enzyme at 50 U / test-200 U / test, and RNase inhibitor at 10 U / test-30 U / test.

[0014] In some embodiments, the PCR amplification composition further includes a lyophilization protectant.

[0015] The freeze-drying protectant comprises one or more of the following: 8 w / v%-12 w / v trehalose, 1 w / v%-3 w / v mannitol, 2 w / v%-5 w / v dextran, and 3 w / v%-6 w / v PEG 6000.

[0016] In some embodiments, the PCR amplification composition includes:

[0017] 10 mM - 30 mM Tris-Cl, pH 8.5; 30 mM - 100 mM KCl; 3 mM - 10 mM MgCl2; 0.5 mM - 2 mM DTT; 0.05 v / v% - 0.5 v / v% Tween-20; 0.3 mM - 0.6 mM dNTP mix; 0.3 mM - 0.5 mM dUTP; 2.2 w / v% - 2.6 w / v% PEG20000; 0.5 mg / mL - 2 mg / mL bovine serum albumin; 0.5 U / test - 2 U / test UDG; 3 U / test - 10 U / test Taq enzyme; 50 U / test - 200 U / test RT enzyme; 10 U / test - 30 U / test RNase inhibitor; 8 w / v% - 12 w / v% trehalose; 1 w / v% - 3 w / v One or more of the following: % mannitol, 2 w / v %-5 w / v % dextran, and 3 w / v %-6 w / v % PEG 6000.

[0018] This application also provides a method for amplifying a target fragment, comprising: mixing a nucleic acid template to be tested with the PCR amplification composition, and performing a PCR amplification reaction.

[0019] In some of these embodiments, the parameters for the PCR amplification reaction include: denaturation temperature of 95℃-96℃, annealing temperature of 58℃-61℃, number of cycles of 35-41, and total amplification time ≤15min.

[0020] In some of these embodiments, the conditions for the PCR amplification reaction include:

[0021] Incubate at 50℃-60℃ for 2-3 minutes; pre-denature at 95℃-96℃ for 10 seconds; denature at 95℃-96℃ for 0-1 seconds; anneal at 58℃-61℃ for 3-8 seconds; repeat 40-41 times.

[0022] This application provides a PCR amplification composition comprising PEG20000 or PEG10000 and a PCR amplification reaction solution used in conjunction with it. This PCR amplification composition utilizes high-molecular-weight PEG as a "molecular congestion agent," increasing the molecular congestion of the reaction system, promoting the binding of DNA polymerase to the template, and improving the enzyme's catalytic efficiency, thereby completing a highly efficient amplification reaction within a shorter denaturation and annealing time. Furthermore, high-molecular-weight PEG can form a stable enzyme-template complex, reducing enzyme denaturation and inactivation during rapid heating and cooling processes, ensuring the specificity and yield of the amplification. Detailed Implementation

[0023] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0024] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0026] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0027] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0028] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0029] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0030] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0031] 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.

[0032] In this application, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions composed of the listed features.

[0033] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0034] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0035] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0036] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0037] Over the past decade, PCR technology has undergone a conceptual leap from an "equilibrium paradigm" to a "kinetic paradigm": no longer pursuing three-stage isothermal cycles, but shifting to a physical model of "continuous temperature variation and instantaneous completion." New platforms such as capillary-hot air, thin-film resistors, microfluidic shuttles, infrared radiation, and water bath stepper motors have pushed heating and cooling rates to 200-400℃ / s, and reduced single-cycle time from min to 2-20 seconds; under extreme conditions, 30 cycles can be completed within 30 seconds, while amplicon specificity and yield are still maintained.

[0038] However, this rapid PCR method has several significant drawbacks: 1) It places extremely high demands on instrument hardware, requiring low-heat-capacity, high-power heating elements (thin-film resistors, infrared lamps, high-speed hot air) and millisecond-level cooling systems (forced air cooling, water bath stepping, Peltier arrays) to work in tandem. This leads to a substantial increase in equipment cost, size, and power consumption. Thermocouples or fiber optic temperature measurements must achieve "zero-delay" matching with the sample; otherwise, temperature overshoot / undershoot will cause enzyme inactivation or non-specific amplification. 2) The system has a weak amplification effect and is difficult to be compatible with conventional consumables. Capillaries, microchambers, or metal tubes can only hold 1-5 µL of reaction solution, and conventional 25-100 µL systems cannot replicate with the same efficiency. 3) Plastic 96-well plates and PCR tubes have high heat capacity and lag in heating and cooling, making it impossible to directly apply second-level programs. 4) The biochemical window is extremely narrow, resulting in low error tolerance. 5) There is no "redundant time" buffer; changes in salt ions, dyes, and excipients can significantly affect specificity and yield. While rapid PCR has demonstrated "extreme speed" in scientific research and prototypes, high hardware requirements, narrow biochemical window, high reagent consumption, and lack of standardization remain the core bottlenecks that must be overcome before it can move from the laboratory to large-scale application.

[0039] Based on this, this application provides a PCR amplification composition, including a PCR amplification reaction solution and PEG10000 or PEG20000.

[0040] The PCR amplification reaction solution includes one or more of the following: 10 mM - 30 mM Tris-Cl, pH 8.5; 30 mM - 100 mM KCl; 3 mM - 10 mM MgCl2; 0.5 mM - 2 mM DTT; 0.05 v / v% - 0.5 v / v% Tween-20; 0.3 mM - 0.6 mM dNTPmix; and 0.3 mM - 0.5 mM dUTP.

[0041] This application provides a PCR amplification composition and a method for introducing high molecular weight polyethylene glycol PEG20000 or PEG10000 as a "macromolecular crowding agent" into the PCR amplification composition. This PEG, by instantaneously increasing the local effective concentration and stabilizing the enzyme-template complex, shortens the conventional 35-41 cycles of RT-qPCR to ≤15 min on a rapid PCR instrument, while maintaining or improving yield and specificity. At this concentration, PEG20000 or PEG10000 can maximally promote PCR amplification, shorten amplification time, and maintain good amplification specificity and yield.

[0042] High molecular weight PEG promotes the binding of DNA polymerase to the template by increasing the molecular crowding of the reaction system, thereby improving the enzyme's catalytic efficiency and enabling efficient amplification reactions to be completed within a shorter denaturation and annealing time. Furthermore, high molecular weight PEG can stabilize the enzyme-template complex, reducing enzyme denaturation and inactivation during rapid heating and cooling processes, ensuring the specificity and yield of amplification.

[0043] This PCR amplification composition can be widely used in various nucleic acid amplification techniques, including but not limited to rapid thermal cycling PCR, rapid qPCR, digital PCR, and isothermal amplification, and has broad application prospects.

[0044] In some embodiments, the final concentration of PEG20000 in the PCR amplification composition is 2.2 w / v%-2.6 w / v%. For example, the final concentration of PEG20000 in the PCR amplification composition is 2.2 w / v%, 2.3 w / v%, 2.4 w / v%, 2.5 w / v%, or 2.6 w / v%, and any value in between.

[0045] In some embodiments, the final concentration of PEG10000 in the PCR amplification composition is 2.2 w / v%-2.6 w / v%. For example, the final concentration of PEG10000 in the PCR amplification composition is 2.2 w / v%, 2.3 w / v%, 2.4 w / v%, 2.5 w / v%, or 2.6 w / v%, and any value in between.

[0046] In some embodiments, the PCR amplification reaction solution also includes bovine serum albumin (BSA). BSA can synergistically work with PEG10000 or PEG20000 to further improve PCR amplification, especially for some difficult-to-amplify targets, showing a significant promoting effect.

[0047] In some embodiments, the final concentration of bovine serum albumin in the PCR amplification composition is 0.5 mg / mL to 2 mg / mL. For example, the final concentration of bovine serum albumin in the PCR amplification composition is 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, or 2.0 mg / mL, and any value in between.

[0048] In some embodiments, the PCR amplification composition further includes an enzyme composition.

[0049] In some embodiments, the enzyme composition comprises one or more of UDG at 0.5 U / test-2 U / test, Taq enzyme at 3 U / test-10 U / test, RT enzyme at 50 U / test-200 U / test, and RNase inhibitor at 10 U / test-30 U / test.

[0050] In some embodiments, the PCR amplification composition further includes a lyophilization protectant.

[0051] The freeze-drying protectant comprises one or more of the following: 8 w / v%-12 w / v trehalose, 1 w / v%-3 w / v mannitol, 2 w / v%-5 w / v dextran, and 3 w / v%-6 w / v PEG 6000. For example, the concentration of trehalose may be 8 w / v%, 9 w / v%, 10 w / v%, 11 w / v%, or 12 w / v, or any value in between.

[0052] For example, mannitol concentrations can be 1 w / v%, 2 w / v%, or 3 w / v%, or any value in between.

[0053] For example, glucose concentrations of 2 w / v%, 3 w / v%, 4 w / v%, or 5 w / v%, or any value in between.

[0054] For example, the concentration of PEG6000 is 3 w / v%, 4 w / v%, 5 w / v%, or 6 w / v%, or any value in between.

[0055] In some embodiments, the PCR amplification composition includes:

[0056] 10 mM - 30 mM Tris-Cl, pH 8.5; 30 mM - 100 mM KCl; 3 mM - 10 mM MgCl2; 0.5 mM - 2 mM DTT; 0.05 v / v% - 0.5 v / v% Tween-20; 0.3 mM - 0.6 mM dNTP mix; 0.3 mM - 0.5 mM dUTP; 2.2 w / v% - 2.6 w / v% PEG20000; 0.5 mg / mL - 2 mg / mL bovine serum albumin; 0.5 U / test - 2 U / test UDG; 3 U / test - 10 U / test Taq enzyme; 50 U / test - 200 U / test RT enzyme; 10 U / test - 30 U / test RNase inhibitor; 8 w / v% - 12 w / v% trehalose; 1 w / v% - 3 w / v One or more of the following: % mannitol, 2 w / v %-5 w / v % dextran, and 3 w / v %-6 w / v % PEG 6000.

[0057] This application also provides a method for amplifying a target fragment, comprising:

[0058] The nucleic acid template to be tested is mixed with the PCR amplification composition, and then PCR amplification is performed. This method, by using a PCR amplification composition containing high molecular weight PEG, enables rapid amplification on conventional PCR instruments without the need for special hardware, greatly lowering the technical threshold for rapid PCR.

[0059] In some of these embodiments, the PCR amplification parameters include: denaturation temperature of 95℃-96℃, annealing temperature of 58℃-61℃, number of cycles of 35-41, and total amplification time ≤15min.

[0060] In some of these embodiments, the PCR amplification parameters include incubation at 50℃-60℃ for 2-3 min; pre-denaturation at 95℃-96℃ for 10 s; denaturation at 95℃-96℃ for 0-1 s; annealing at 58℃-61℃ for 3-8 s; and 40-41 cycles.

[0061] This application covers the use of the PCR amplification composition in rapid thermal cycling PCR, rapid qPCR, digital PCR, and isothermal amplification, as well as ready-to-use premixes and matching kits in lyophilized systems. Compared with conventional PCR amplification compositions, it significantly shortens the detection time and improves detection efficiency while maintaining the same detection rate.

[0062] 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 specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0063] In the specific embodiments described below, the measurement parameters involving 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.

[0064] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0065] Example 1: Screening of PEGs with different molecular weights

[0066] 1. Materials and Methods:

[0067] Sample: Nucleic acid reference panel for seven respiratory pathogens (prepared by Sansure Biotech);

[0068] Amplification instrument: Slan48SC (Hongshi).

[0069] Reagents: Seven respiratory pathogen detection kit (batch number Y25003-J), cell preservation solution (X1010) (production batch number: 25001).

[0070] 2. Sample preparation

[0071] (1) Dilute reference sample S1 in the seven respiratory pathogen nucleic acid reference disk with cell preservation solution (X1010) (production batch number: 25001) to 1000 copies / mL.

[0072] 3. Experimental Procedure

[0073] (1) Add 22 μL of template diluted to 1000 copies / mL to each PCR amplification tube containing lyophilized bulbs, and then add the following additives to a total volume of 25 μL:

[0074] Example 1-1: Add 3 μL of 20% PEG20000 (final concentration 2.4%).

[0075] Examples 1-2: Add 3 μL of 20% PEG10000 (final concentration 2.4%).

[0076] Comparative Example 1-1: Add 3 μL of deionized H2O (without additives).

[0077] Comparative Examples 1-2: Add 3 μL of 20% PEG8000 (final concentration 2.4%).

[0078] Comparative Examples 1-3: Add 3 μL of 20% PEG4000 (final concentration 2.4%).

[0079] Comparative Examples 1-4: Add 3 μL of 20% PEG2000 (final concentration 2.4%).

[0080] Comparative Examples 1-5: Add 2 μL of 20% PEG20000 (final concentration 1.6%) and 1 μL of PEG600 (final concentration 4%).

[0081] Apart from the additives, the PCR amplification composition consists of: 20 mM Tris-Cl, pH 8.5, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1 v / v% Tween-20, 0.5 mM dNTP mix, 0.5 mM dUTP, 1 U / test UDG, 5 U / test Taq enzyme, 100 U / test RT enzyme, 20 U / test RNase inhibitor, 10 w / v% trehalose, 2 w / v% mannitol, 3 w / v% dextran, and 5 w / v% PEG 6000.

[0082] (2) Shake well, centrifuge, and then perform PCR amplification.

[0083] The details are shown in Table 1 below:

[0084] Table 1

[0085]

[0086] 4. Amplification program

[0087] (1) The rapid amplification procedure is shown in Table 2 below:

[0088] Table 2

[0089]

[0090] The program takes 18 minutes to run on the Slan48SC amplification instrument and 15 minutes to run on the SureXVI amplification instrument.

[0091] 5. The experimental results are shown in Table 3 below:

[0092] Table 3

[0093]

[0094] Conclusion: Based on the results analysis, under the same rapid amplification program conditions, PEG20000 and PEG10000 showed more significant effects, advancing the CT value by 1-3 points. In contrast, PEG8000 (Comparative Examples 1-2), PEG4000 (Comparative Examples 1-3), and PEG2000 (Comparative Examples 1-4) had virtually no promoting effect. In Comparative Examples 1-5, PEG20000 and PEG600 likely played a major role. Therefore, PEG10000 and PEG20000 are the preferred molecular blocking agents, and PEG20000 will be used for subsequent validation.

[0095] Example 2: Comparison of PEG20000 with other types of congestion-prone agents

[0096] 1. Materials and Methods:

[0097] Sample: Enterprise reference sample (made by Sansure Biotech).

[0098] Amplification instrument: Slan48SC (Hongshi).

[0099] Reagents: Seven respiratory pathogen detection kit (batch number Y25003-J), cell preservation solution (X1010) (production batch number: 25001).

[0100] 2. Sample preparation

[0101] (1) Dilute reference sample S1 in the respiratory pathogen nucleic acid reference plate to 1000 copies / mL with cell preservation solution (X1010).

[0102] 3. Experimental Procedure

[0103] (1) Add 22 μL of template diluted to 1000 copies / mL to each PCR amplification tube containing lyophilized bulbs, and then add the following additives to a total volume of 25 μL:

[0104] Example 2-1: Add 3 μL of 20% PEG20000 (final concentration 2.4%).

[0105] Comparative Example 2-1: Add 3 μL of deionized H2O (without additives).

[0106] Comparative Example 2-2: Add 3 μL of 20% sucrose (final concentration 2.4%).

[0107] Comparative Examples 2-3: Add 3 μL of 1 M trehalose (final concentration 120 mM).

[0108] Apart from the additives, the PCR amplification composition consists of: 20 mM Tris-Cl, pH 8.5, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1 v / v% Tween-20, 0.5 mM dNTP mix, 0.5 mM dUTP, 1 U / test UDG, 5 U / test Taq enzyme, 100 U / test RT enzyme, 20 U / test RNase inhibitor, 10 w / v% trehalose, 2 w / v% mannitol, 3 w / v% dextran, and 5 w / v% PEG 6000.

[0109] (2) Shake well, centrifuge, and then perform PCR amplification.

[0110] The results are shown in Table 4:

[0111] Table 4: Experiment Setup and Instructions

[0112]

[0113] 4. The amplification procedure is shown in Table 5 below:

[0114] Table 5: Amplification Procedure

[0115]

[0116] 5. The experimental results are shown in Table 6 below:

[0117] Table 6: Comparison of different congestion agents in rapid amplification programs

[0118]

[0119] Conclusion: Based on the results analysis, the Ct value of Example 2-1 (PEG20000) was significantly lower than that of Comparative Example 2-1 (no additive), Comparative Example 2-2 (sucrose) and Comparative Example 2-3 (trehalose), indicating that PEG20000 has a unique promoting effect in this system, while sucrose and trehalose are not suitable.

[0120] Example 3: Screening of PEG concentration

[0121] 1. Materials and Methods:

[0122] Sample: Enterprise reference sample (made by Sansure Biotech).

[0123] Amplification instrument: Slan48SC (Hongshi).

[0124] Reagents: Seven respiratory pathogen detection kit (batch number Y25003-J), cell preservation solution (X1010) (production batch number: 25001).

[0125] 2. Sample preparation

[0126] (1) Dilute reference sample S1 in the respiratory pathogen nucleic acid reference plate to 1000 copies / mL with cell preservation solution (X1010).

[0127] 3. Experimental Procedure

[0128] (1) Add 22 μL of template diluted to 1000 copies / mL to each PCR amplification tube containing lyophilized pellets, then add different volumes of 20% PEG20000 (0-5 μL), and make up to a total volume of 25 μL with deionized H2O. The final concentration range of PEG20000 is 0-4%.

[0129] Comparative Example 3-1: Add 0 μL of PEG20000 (final concentration 0%).

[0130] Example 3-1: Add 1 μL of PEG20000 (final concentration 0.8%).

[0131] Example 3-2: Add 1.5 μL of PEG20000 (final concentration 1.2%).

[0132] Example 3-3: Add 2 μL of PEG20000 (final concentration 1.6%).

[0133] Examples 3-4: Add 2.5 μL of PEG20000 (final concentration 2.0%).

[0134] Examples 3-5: Add 3 μL of PEG20000 (final concentration 2.4%).

[0135] Examples 3-6: Add 3.5 μL of PEG20000 (final concentration 2.8%).

[0136] Examples 3-7: Add 4 μL of PEG20000 (final concentration 3.2%).

[0137] Examples 3-8: Add 5 μL of PEG20000 (final concentration 4%).

[0138] Apart from the additives, the PCR amplification composition consists of: 20 mM Tris-Cl, pH 8.5, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1 v / v% Tween-20, 0.5 mM dNTP mix, 0.5 mM dUTP, 1 U / test UDG, 5 U / test Taq enzyme, 100 U / test RT enzyme, 20 U / test RNase inhibitor, 10 w / v% trehalose, 2 w / v% mannitol, 3 w / v% dextran, and 5 w / v% PEG 6000.

[0139] (2) After vortexing and mixing, centrifuge, and then perform PCR amplification. 4. The amplification program is shown in Table 7 below:

[0140] Table 7: Amplification Procedure

[0141]

[0142] The program ran for 14 minutes and 50 seconds on the Slan48SC (Hongshi) amplification instrument.

[0143] 4. The experimental results are shown in Table 8 below:

[0144] Table 8: Application of different concentrations of PEG20000 in rapid amplification programs

[0145]

[0146] Conclusion: Data analysis showed that PEG20000 improved amplification performance within a final concentration range of 0.8%-4%, but Examples 3-5 (final concentration 2.4%) exhibited the lowest Ct values ​​and the best amplification performance. Therefore, it is preferable to add 3 μL of 20% PEG20000 (final concentration 2.4%) to a 25 μL amplification system.

[0147] Example 4: Combination and Optimization with Other Reagents

[0148] 1. Materials and Methods:

[0149] Sample: Enterprise reference material (self-made by Sansure Biotech) Amplification instrument: Slan48SC (Hongshi).

[0150] Reagents: Seven respiratory pathogen detection kit (Sansure Biotech, batch number Y25003-J), cell preservation solution (X1010) (Sansure Biotech, batch number: 25001).

[0151] 2. Sample preparation

[0152] The reference sample S1 in the respiratory pathogen nucleic acid reference disk was diluted to 1000 copies / mL with cell preservation solution (X1010).

[0153] 3. Experimental Procedure

[0154] (1) Add 20 μL of template diluted to 1000 copies / mL to each PCR amplification tube containing lyophilized pellets, and add other tubes with different additives or deionized H2O to bring the total volume to 25 μL.

[0155] Comparative Example 4-1: Add 5 μL of deionized H2O (without additives).

[0156] Example 4-1: Add 3 μL of 20% PEG20000 (final concentration 2.4%) and 2 μL of H2O.

[0157] Example 4-2: Add 3 μL of 20% PEG20000 (final concentration 2.4%), 0.5 μL of 50 mg / mL BSA (final concentration 1 mg / mL) and 1.5 μL of H2O.

[0158] Example 4-3: Add 3 μL of 20% PEG20000 (final concentration 2.4%), 1 μL of 50 mg / mL BSA (final concentration 2 mg / mL) and 1 μL of H2O.

[0159] Example 4-4: Add 3 μL of 20% PEG20000 (final concentration 2.4%), 0.5 μL of 50 mg / mL BSA (final concentration 1 mg / mL), 0.5 μL of H2O and 1 μL of 20% Tween-20 (final concentration 0.8%).

[0160] Examples 4-5: Add 3 μL of 20% PEG20000 (final concentration 2.4%), 0.5 μL of 50 mg / mL BSA (final concentration 1 mg / mL), 0.5 μL of H2O and 1 μL of 20% NP-40 (final concentration 0.8%).

[0161] The details are shown in Table 9 below:

[0162] Table 9

[0163]

[0164] Apart from the additives, the PCR amplification composition consists of: 20 mM Tris-Cl, pH 8.5, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1 v / v% Tween-20, 0.5 mM dNTP mix, 0.5 mM dUTP, 1 U / test UDG, 5 U / test Taq enzyme, 100 U / test RT enzyme, 20 U / test RNase inhibitor, 10 w / v% trehalose, 2 w / v% mannitol, 3 w / v% dextran, and 5 w / v% PEG 6000.

[0165] (2) After shaking to mix, centrifuge and then perform PCR amplification.

[0166] (3) The amplification procedure is shown in Table 10 below:

[0167] Table 10

[0168]

[0169] The program ran for 14 minutes and 50 seconds on the Slan48SC (Hongshi) amplification instrument.

[0170] 4. The experimental results are shown in Table 11 below:

[0171] Table 11

[0172]

[0173] Conclusion: The Ct values ​​of Example 4-3 (PEG20000 and BSA combination) were low in multiple channels, indicating that BSA could further improve the amplification effect. Examples 4-4 and 4-5 (with surfactant added) did not show significant advantages. Therefore, a 25 μL amplification system containing a final concentration of 2.4% PEG20000 and 2 mg / mL BSA was selected.

[0174] Example 5: Exploration of an ultrafast amplification program based on the proposed amplification composition

[0175] 1. Materials and Methods:

[0176] Samples: Clinical samples of respiratory pathogens (Fuzhou Children's Hospital).

[0177] Amplification instrument: SureXVI (Sansure Biotech) amplification instrument.

[0178] Reagent: Seven respiratory pathogen detection kit (Sansure Biotech, batch number Y25003-J).

[0179] Cell preservation solution (X1010) (Sansure Biotech, production batch number: 25001).

[0180] 2. Sample preparation

[0181] (1) After mixing high concentrations of clinical samples of respiratory pathogens, dilute 50 times with cell preservation solution (X1010) and add swabs from healthy individuals.

[0182] 3. Experimental Procedure

[0183] Add 21.5 μL of the processed clinical sample nucleic acid to a PCR tube and set up the following two groups:

[0184] Comparative Example 5-1 (Control Group): Add 3.5 μL of deionized water (using the original system of the kit).

[0185] Example 5-1 (Experimental group, composition of this application): 3 μL of 20% PEG20000 and 0.5 μL of 50 mg / mL BSA were added.

[0186] The two groups of samples were tested using four different amplification programs with varying runtimes, as shown in the table below. Amplification program 1 (18 min) was used as the benchmark for performance comparison.

[0187] Apart from the additives, the PCR amplification composition consists of: 20 mM Tris-Cl, pH 8.5, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1 v / v% Tween-20, 0.5 mM dNTP mix, 0.5 mM dUTP, 1 U / test UDG, 5 U / test Taq enzyme, 100 U / test RT enzyme, 20 U / test RNase inhibitor, 10 w / v% trehalose, 2 w / v% mannitol, 3 w / v% dextran, and 5 w / v% PEG 6000.

[0188] 4. The amplification procedure is shown in Table 12 below:

[0189] Table 12

[0190]

[0191] 5. The experimental results are shown in Table 13 below:

[0192] Table 13

[0193]

[0194] Conclusion: Accelerated program: Using the composition of this application, the amplification program can be successfully shortened from 18 min (program 1) to 13-15 min (programs 2, 3, 4), achieving ultra-fast detection.

[0195] Guaranteed detection performance: Under a 15-minute program, the detection Ct values ​​of the composition of this application (FAM: 30.28; HEX: 31.58) are even superior to those of conventional systems under an 18-minute program (FAM: 31.02; HEX: 31.72). Even under the shortest 13-minute program, its detection performance (Ct value) is still comparable to that of the conventional 18-minute program.

[0196] Example 6: Application of the PCR amplification composition of this application in ultra-rapid and highly sensitive detection of Mycobacterium tuberculosis.

[0197] 1. Materials and Methods: Materials: Mycobacterium tuberculosis (Zopf) Lehmann and Neumann H37Ra quality control product CBPK0064 (i.e., Mycobacterium tuberculosis standard, Kebai); 5× buffer (Kangde, TB202504); enzyme MIX (Kangde, TB202504); cell preservation solution (X1010) (Sansure Biotech, production batch number: 25001).

[0198] 2. Sample preparation

[0199] (1) Sample: Mycobacterium tuberculosis (Zopf) Lehmann and Neumann H37Ra quality control product CBPK0064 (also known as Mycobacterium tuberculosis standard, Kebai).

[0200] (2) Instruments: SureXVI (Hongshi) amplification instrument. Ultrasound instrument (Tienan SS-8S, or Xinzhi Bio SS-8).

[0201] 3. Method: Cell preservation solution (X1010) (Sansure Biotech, production batch number: 25001).

[0202] The Mycobacterium tuberculosis standard was diluted to 10 CFU / mL and 1 CFU / mL, and tongue swabs from healthy individuals were added. The mixture was then sonicated for 2 min to prepare the nucleic acid sample for testing. 21.5 μL of the treated nucleic acid sample was added to a PCR amplification tube, and the following two groups were set up for comparison:

[0203] Comparative Example 6-1 (Control Group, Standard System): Add 3.5 μL of deionized water.

[0204] Example 6-1 (Experimental group, system of this application): 3 μL of 20% PEG20000 (final concentration 2.4%) and 0.5 μL of 50 mg / mL BSA (final concentration 1 mg / mL) were added.

[0205] Then, perform amplification detection according to the amplification procedure shown in Table 14-15 and compare the CT values.

[0206] After vortexing and mixing, centrifuge, and then perform PCR amplification.

[0207] 4. Amplification program

[0208] Table 14: Amplification Program 1 (Running time in SureXVI: 39 min)

[0209]

[0210] Table 15: Amplification Program 2 (Running time in SureXVI: 18 min)

[0211]

[0212] 5. The experimental results are shown in Table 16 below:

[0213] Table 16

[0214]

[0215] 6. Conclusion: Based on the results analysis, for a sample of 10 CFU / mL, the detection effect of the composition of this application under the ultra-fast program of 18 min (Ct=31.69) is comparable to that of the conventional system under the program of 39 min (Ct=30.89), successfully reducing the detection time by more than half.

[0216] For samples with extremely low concentrations of 1 CFU / mL that are even more difficult to detect, the conventional system is essentially ineffective under the rapid program (Ct=38.68). However, when using the composition of this application, under the same 18-minute rapid program, the Ct value (34.40) is comparable to that of the 39-minute conventional program (34.02), and the Ct value is achieved more than 4 cycles earlier.

[0217] The results above demonstrate that adding PEG20000 and BSA can significantly shorten the amplification time, and the amplification is significantly improved compared with the control under the same amplification program conditions, especially for low concentration samples.

[0218] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. 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 scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A PCR amplification composition, characterized in that, Includes PCR amplification reaction solution and PEG20000; The PCR amplification reaction solution includes: 10 mM-30 mM Tris-Cl, pH 8.5, 30 mM-100 mM KCl, 3 mM-10 mM MgCl2, 0.5 mM-2 mM DTT, 0.05 v / v%-0.5 v / v% Tween-20, 0.3 mM-0.6 mM dNTP mix, and 0.3 mM-0.5 mM dUTP; The final concentration of PEG20000 in the PCR amplification composition is 2.2 w / v%-2.6 w / v%. The PCR amplification reaction solution also includes bovine serum albumin; The final concentration of bovine serum albumin in the PCR amplification composition is 0.5 mg / mL to 2 mg / mL.

2. The PCR amplification composition according to claim 1, characterized in that, The PCR amplification composition also includes an enzyme composition.

3. The PCR amplification composition according to claim 2, characterized in that, The enzyme composition comprises one or more of the following: 0.5 U / test-2 U / test of UDG, 3 U / test-10 U / test of Taq enzyme, 50 U / test-200 U / test of RT enzyme, and 10 U / test-30 U / test of RNase inhibitor.

4. The PCR amplification composition according to claim 3, characterized in that, The PCR amplification composition also includes a lyophilization protectant; The freeze-drying protectant includes one or more of the following: 8 w / v%-12 w / v trehalose, 1 w / v%-3 w / v mannitol, 2 w / v%-5 w / v dextran, and 3 w / v%-6 w / v PEG 6000.

Images