Fluorescence / chromogenic dual-mode lamp detection reagent, preparation method and application thereof

Abstract

This invention belongs to the field of LAMP detection reagent technology, specifically relating to a fluorescence / colorimetric dual-mode LAMP detection reagent, its preparation method, and its application. The fluorescence / colorimetric dual-mode LAMP detection reagent of this invention comprises: BST DNA polymerase, magnesium sulfate, potassium chloride, phenol red, BSA, mannitol, trehalose, a mixture of dNTPs, a DNA-binding fluorescent dye, and ammonium sulfate. This fluorescence / colorimetric dual-mode LAMP detection reagent of the present invention simultaneously contains a phenol red colorimetric system and a SYBR Green I fluorescence system, allowing the same reaction tube to be read using a fluorescence instrument or observed visually. It eliminates the need to pre-select reagent types based on the detection scenario, providing the flexibility of "one tube, two modes" to adapt to more diverse detection needs. By introducing ammonium sulfate as a reaction enhancer, the different requirements of the two reading modes for the ionic environment are balanced, achieving "dual excellence" in both fluorescence signal and colorimetric effect.

Description

Technical Field

[0001] This invention belongs to the field of LAMP detection reagent technology, specifically relating to a fluorescence / colorimetric dual-mode LAMP detection reagent, its preparation method, and its application. Background Technology

[0002] LAMP technology, due to its isothermal amplification, rapid and efficient characteristics, is widely used in field pathogen detection. The interpretation of detection results mainly falls into two categories: Firstly, there are fluorescence methods (such as SYBR Green I and EvaGreen), which require a quantitative real-time PCR instrument or UV lamp for objective and quantitative results. For example, the core components of existing fluorescence-specific LAMP premixes (such as the NEB Luna Universal Probe One-Step RT-LAMP Kit) include: BST enzyme, fluorescent dye (such as SYBR Green I or EvaGreen), Tris buffer, magnesium sulfate, potassium chloride, and dNTPs. These reagents can only be read using fluorescence equipment, lack color development capabilities, and are incompatible with phenol red.

[0003] Secondly, there are colorimetric methods (such as phenol red), which can be directly interpreted by the naked eye and are suitable for resource-scarce scenarios. For example, the core components of existing colorimetric LAMP premixes (such as NEB WarmStart Colorimetric LAMP 2× Master Mix) are: BST enzyme, phenol red, Tris buffer, magnesium sulfate, potassium chloride, dNTPs, and betaine. This type of reagent can only be interpreted by the naked eye, is not compatible with fluorescence equipment, and does not contain fluorescent dyes.

[0004] While both interpretation methods of the test results have their advantages, existing LAMP assay products cannot simultaneously utilize fluorescence and colorimetric methods in the same reagent. They are either dedicated to fluorescence or colorimetric methods, requiring users to choose in advance based on their equipment, thus failing to achieve the flexibility of a "dual-mode" solution. Furthermore, SYBR Green I is easily degraded during lyophilization, and existing lyophilized fluorescent products generally have a short shelf life (typically ≤6 months) and exhibit significant signal attenuation. Directly adding SYBR Green I to the phenol red system leads to fluorescence signal quenching and colorimetric threshold drift, preventing the two from working synergistically. Colorimetric reagents are incompatible with fluorescence equipment, and fluorescence reagents cannot be visually interpreted, limiting the flexibility of the testing scenarios. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescence / colorimetric dual-mode LAMP detection reagent, its preparation method, and its application.

[0006] The first aspect of this application provides a fluorescence / colorimetric dual-mode LAMP detection reagent, comprising: BST DNA polymerase, magnesium sulfate, potassium chloride, phenol red, BSA, mannitol, trehalose, a mixture of dNTPs, a DNA-binding fluorescent dye, and ammonium sulfate.

[0007] In one embodiment of this application, the concentrations of each component in the 2× working concentration detection reagent are: BST DNA polymerase 0.4-1.2 U / μL; Magnesium sulfate 4-12 mM; Potassium chloride 10-50 mM; Phenol red 50-200 μM; BSA 0.1-0.5 mg / mL; Mannitol 1%-5% (m / v); Trehalose 2%-8% (m / v); dNTPs mixture 0.6-2.0 mM; DNA-binding fluorescent dye, 10-50 μM; Ammonium sulfate 10-40mM.

[0008] In one embodiment of this application, the concentrations of each component in the 2× working concentration detection reagent are: BST DNA polymerase 0.8 U / μL; Magnesium sulfate 8 mM; Potassium chloride 20 mM; Phenol red 100 μM; BSA 0.2 mg / mL; Mannitol 3% (m / v); Trehalose 5% (m / v); dNTPs mixture 1.4 mM; 20 μM DNA-binding fluorescent dye; Ammonium sulfate 25mM.

[0009] In one embodiment of this application, at least one of the following features is included: The DNA-binding fluorescent dyes include one of the following: SYBR Green I, EvaGreen, SYTO-9, and LC Green Plus; The ammonium sulfate can be partially replaced by other ammonium salts such as ammonium acetate and ammonium chloride; The trehalose may be completely or partially replaced with sucrose; for example, it may be partially replaced at a ratio of 1:0.5 to 1:1. The mannitol may be replaced in whole or in part with sorbitol; for example, it may be partially replaced in a ratio of 1:0.5 to 1:1. The potassium chloride is replaced with potassium acetate; a 1:1 replacement ratio is acceptable. The magnesium sulfate is replaced with magnesium chloride; the ratio can be 1:0.8.

[0010] In one embodiment of this application, the 2× working concentration detection reagent further includes 0.2-0.8 U / μL of reverse transcriptase.

[0011] In one embodiment of this application, the 2× working concentration detection reagent also includes 0.6 U / μL of reverse transcriptase.

[0012] A second aspect of this application provides a method for preparing the fluorescence / colorimetric dual-mode LAMP detection reagent as described above, comprising: Preparation environment: Operate in an ice pack at 2-8℃, and keep away from light throughout the process; Add the following ingredients to the enzyme-free centrifuge tube in sequence: deionized water, potassium chloride, magnesium sulfate, ammonium sulfate, dNTPs mixture, phenol red, BSA, mannitol, and trehalose, and mix well; Fine-tune the initial pH to 8.6-8.8 using dilute hydrochloric acid / sodium hydroxide; Add SYBR Green I under light-protected conditions and mix well; Finally, add BST DNA polymerase and mix well. After passing quality inspection, the reagents are packaged in the dark to obtain fresh liquid test reagents.

[0013] A third aspect of this application provides the application of the fluorescence / colorimetric dual-mode LAMP detection reagent described above in fluorescence and / or colorimetric LAMP nucleic acid detection.

[0014] A fourth aspect of this application provides a lyophilized formulation, comprising: a formulation formed by lyophilizing a liquid detection reagent and a LAMP primer set at a working concentration; wherein... The liquid detection reagent is the fluorescence / colorimetric dual-mode LAMP detection reagent as described above.

[0015] The fifth aspect of this application provides the use of the lyophilized formulation described above in fluorescence and / or colorimetric LAMP nucleic acid detection.

[0016] The beneficial effects of this invention are: The fluorescence / colorimetric dual-mode LAMP detection reagent of this invention simultaneously comprises a phenol red colorimetric system and a SYBR Green I fluorescence system, allowing the same reaction tube to be read using a fluorescence instrument or observed visually. It eliminates the need to pre-select reagent types based on the detection scenario, providing the flexibility of "one tube, two modes" to adapt to a wider range of detection needs. By introducing ammonium sulfate as a reaction enhancer, the different requirements of the two reading modes on the ionic environment are balanced, achieving a "dual advantage" in both fluorescence signal and colorimetric effect.

[0017] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the colorimetric effect after the LAMP reaction with reagent C; in the diagram, red represents negative and yellow represents positive. Figure 2 This is a schematic diagram comparing the LAMP reaction of reagents from group A and group C under a UV lamp; in the diagram, the left side shows that the colorimetric reagent from group A has no fluorescence, while the right side shows that the reagent from group C has fluorescence. Figure 3 This is a comparison curve output on a real-time PCR instrument after the LAMP reaction of reagents from group A and group C; in the figure, green represents reagents from group A and red represents reagents from group C. Figure 4 This is a comparison curve output on a real-time PCR instrument after the LAMP reaction of reagents from group A, group B, group C, and group D. In the figure, green represents reagent A, red represents reagent B, yellow represents reagent C, and blue represents reagent D. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This application provides a fluorescence / colorimetric dual-mode LAMP detection reagent, its preparation method, and its application, which are described in detail below. It should be noted that the order of description in the following embodiments is not intended to limit the preferred order of the embodiments of this application. Furthermore, in the following embodiments, the descriptions of each embodiment have their own emphasis; parts not described in detail in a certain embodiment can be referred to in the relevant descriptions of other embodiments.

[0023] An embodiment of the present invention provides a fluorescence / colorimetric dual-mode LAMP detection reagent, comprising: BST DNA polymerase, magnesium sulfate, potassium chloride, phenol red, BSA, mannitol, trehalose, a mixture of dNTPs, SYBR Green I, and ammonium sulfate.

[0024] Specifically, in a 2× working concentration test reagent, the concentrations of each component are as follows: BST DNA polymerase 0.4-1.2 U / μL, preferably 0.8 U / μL; it is the core amplification enzyme; Magnesium sulfate 4-12 mM, preferably 8 mM; used to provide magnesium ions and maintain ionic strength and initial pH stability; Potassium chloride 10-50 mM, preferably 20 mM, is used to match the optimal ionic environment of BST DNA polymerase and adjust the initial pH. Phenol red 50-200 μM, preferably 100 μM; as a pH-sensitive colorimetric reagent, it can achieve a precipitous red-yellow color change for visual interpretation. BSA (bovine serum albumin) 0.1-0.5 mg / mL, preferably 0.2 mg / mL; it is an enzyme protectant, also known as "phenol red chaperone protein"; Mannitol 1%-5% (m / v), preferably 3% (m / v); it is a lyophilization excipient that stabilizes the pH of the microenvironment; Trehalose 2%-8% (m / v), preferably 5% (m / v); it is the core freeze-drying protectant, forming a glassy encapsulation of enzymes and primers; The dNTPs mixture is 0.6-2.0 mM, preferably 1.4 mM; it serves as a raw material for nucleic acid amplification. SYBR Green I 10-50 μM, preferably 20 μM; used for fluorescence interpretation; Ammonium sulfate 10-40mM, preferably 25mM; as a reaction synergist, it balances the ionic compatibility of SYBR and phenol red, and synergistically protects against lyophilization.

[0025] It should be noted that commercially available SYBR GREEN I is often a high-expansion mixed solution. The concentration of SYBR Green I in the 2× working concentration test reagent here is calculated based on the actual amount of SYBR Green I added in the high-expansion mixed solution. Other trace components in commercially available mixed solutions only have a stabilizing and solubilizing effect on SYBR GREEN I and have no effect on the reaction.

[0026] An embodiment of this application also provides a method for preparing the fluorescence / colorimetric dual-mode LAMP detection reagent as described above, comprising: Preparation environment: Operate in an ice pack at 2-8℃, and keep away from light throughout the process; Add the following ingredients to the enzyme-free centrifuge tube in sequence: deionized water, potassium chloride, magnesium sulfate, ammonium sulfate, dNTPs mixture, phenol red, BSA, mannitol, and trehalose, and mix well; Fine-tune the initial pH to 8.6-8.8 using dilute hydrochloric acid / sodium hydroxide; Add SYBR Green I under light-protected conditions and mix well; Finally, add BST DNA polymerase and mix well. After passing quality inspection, the reagents are packaged in the dark to obtain fresh liquid test reagents.

[0027] One embodiment of this application also provides a lyophilized formulation, comprising: a formulation formed by lyophilizing a liquid detection reagent and a LAMP primer set at a working concentration; wherein... The liquid detection reagent is the fluorescence / colorimetric dual-mode LAMP detection reagent as described above.

[0028] Specifically, the lyophilized formulation can be prepared as follows: Mix the above-mentioned fresh liquid detection reagents with the LAMP primer set at the working concentration, and keep the mixture away from light throughout the process; Aliquot the product into PCR tubes according to the single reaction volume (≤50 μL / tube). Freeze-drying (avoiding light throughout the process): Pre-freezing: Pre-freeze at -40℃ for 4 hours; One-time drying: Vacuum degree 10 Pa, sublimation at -30℃ for 12 hours; Secondary drying: vacuum degree 5 Pa, 25℃ for 3 hours; After freeze-drying, the product is directly capped and sealed under vacuum to obtain a dual-mode integrated freeze-dried formulation.

[0029] In some embodiments, the freeze-drying parameters can be adjusted according to the equipment performance, with a pre-freezing temperature of -35°C to -50°C and a total freeze-drying time of 16-24 hours.

[0030] The detection reagent of this invention can be used in LAMP nucleic acid detection scenarios, and users can freely choose the interpretation method according to their equipment. Real-time quantitative PCR instrument: can monitor the amplification curve in real time and perform quantitative analysis; Ultraviolet lamp / blue light transilluminator: can observe fluorescence signals and perform qualitative interpretation; Direct visual observation: The results can be directly interpreted based on the changes in red and yellow colors.

[0031] Experiment 1: Dual-mode compatibility verification Grouping of test reagents: Group A: Phenol red colorimetric system only (control) Group B: SYBR Green I fluorescence system only (control) Group C: Contains phenol red + SYBR + ammonium sulfate (this invention) Group D: Contains phenol red + SYBR, without ammonium sulfate (control). Group formulation

[0032] II. Primer Sequences and Formulations

[0033] III. Freeze-drying system formulation

[0034] Methods: The reagents and primers of each group were mixed and lyophilized to form lyophilized formulations; 10 3 Using copies / μL of E. coli genomic DNA as a template (genomic DNA extracted from E. coli, kit product of Shanghai Sangon Biotech, catalog number: B518225-0050), amplification was performed at 63℃ for 30 minutes. The Ct value was detected by a real-time PCR instrument, and the color development results were observed visually.

[0035] result: The fluorescence signal intensity of group C was not significantly different from that of group B, and the color development effect was not significantly different from that of group A. Group D showed fluorescence signal attenuation (signal intensity decrease >50%) or sluggish color development (yellowing time delay >5 minutes). This demonstrates that ammonium sulfate is a key component for achieving dual-mode compatibility.

[0036] Experiment 2: Stability of fluorescence signal before and after freeze-drying Methods: The lyophilized formulation of Experiment 1C was stored at 25℃ in the dark for 0, 1, 3, and 6 months, then reconstituted and subjected to LAMP reaction. The reaction was carried out using a 10-fold LAMP reaction. 3 Using copies / μL of E. coli genomic DNA as a template (genomic DNA extracted from E. coli, kit product of Shanghai Sangon Biotech, catalog number: B518225-0050), amplification was performed at 63℃ for 30 minutes, and the fluorescence signal intensity was detected and compared with that of fresh liquid preparation.

[0037] result: After 6 months, the fluorescence signal retention rate was ≥80%; No fluorescence quenching was observed after lyophilization and reconstitution, and the signal was uniform.

[0038] The core principle of the detection reagent of this invention is as follows: (1) The "dual action" mechanism of ammonium sulfate By introducing ammonium sulfate, the following synergistic effects are achieved: a. Balanced ion compatibility: SYBR Green I is compatible with Mg 2+ Concentration-sensitive, the addition of ammonium sulfate can adjust the ionic strength window, allowing SYBR Green I to maintain the optimal fluorescence quantum yield in the phenol red system without interfering with the colorimetric threshold of phenol red; b. Synergistic freeze-drying protection: Ammonium sulfate forms a complex protective network with trehalose and mannitol, which stabilizes the molecular conformation of SYBR Green I during freeze-drying and prevents its aggregation and degradation; c. Enhanced amplification efficiency: Ammonium sulfate, as a traditional LAMP enhancer, can improve the amplification efficiency of BST enzyme and shorten the peak time.

[0039] (2) Design of dual-mode compatible ion environment By precisely controlling the ratio of magnesium sulfate, potassium chloride, and ammonium sulfate, an ionic environment was constructed that optimally satisfies both the SYBR GreenI fluorescence signal and ensures a clear phenol red colorimetric threshold. Under this ionic environment: The fluorescence signal intensity of SYBR Green I is comparable to that of dedicated fluorescent reagents; The phenol red color development window does not drift and can still clearly turn yellow after positive amplification; The results of fluorescence detection were 100% consistent with those of visual interpretation.

[0040] It should be noted that all the devices (parts whose specific structures are not specified) selected in this application are general standard parts or parts known to those skilled in the art, and their structures and principles can be known to those skilled in the art through technical manuals or conventional experimental methods.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A fluorescent / chromogenic dual mode LAMP detection reagent, characterized in that, include: BST DNA polymerase, magnesium sulfate, potassium chloride, phenol red, BSA, mannitol, trehalose, dNTPs mixture, DNA-binding fluorescent dye, ammonium sulfate.

2. The detection reagent according to claim 1, characterized by In a 2× working concentration test reagent, the concentrations of each component are: BST DNA polymerase 0.4-1.2 U / μL; Magnesium sulfate 4-12 mM; Potassium chloride 10-50 mM; Phenol red 50-200 μM; BSA 0.1-0.5 mg / mL; Mannitol 1%-5% (m / v); Trehalose 2%-8% (m / v); dNTPs mixture 0.6-2.0 mM; DNA-binding fluorescent dye, 10-50 μM; Ammonium sulfate 10-40mM.

3. The detection reagent according to claim 2, characterized in that, In a 2× working concentration test reagent, the concentrations of each component are: BST DNA polymerase 0.8 U / μL; Magnesium sulfate 8 mM; Potassium chloride 20 mM; Phenol red 100 μM; BSA 0.2 mg / mL; Mannitol 3% (m / v); Trehalose 5% (m / v); dNTPs mixture 1.4 mM; 20 μM DNA-binding fluorescent dye; Ammonium sulfate 25mM.

4. The detection reagent according to claim 2, characterized by Includes at least one of the following features: The DNA-binding fluorescent dyes include one of the following: SYBR Green I, EvaGreen, SYTO-9, and LC Green Plus; The ammonium sulfate is partially replaced with ammonium acetate or ammonium chloride; The trehalose may be entirely or partially replaced with sucrose; The mannitol was replaced in whole or in part with sorbitol; The potassium chloride is replaced with potassium acetate; The magnesium sulfate was replaced with magnesium chloride.

5. The detection reagent according to claim 1, characterized by The 2× working concentration test reagent also includes 0.2-0.8 U / μL of reverse transcriptase.

6. The detection reagent according to claim 5, characterized in that, The 2× working concentration test reagent also includes 0.6 U / μL of reverse transcriptase.

7. A method for preparing a fluorescence / colorimetric dual-mode LAMP detection reagent as described in any one of claims 1-6, characterized in that, include: Preparation environment: Operate in an ice pack at 2-8℃, and keep away from light throughout the process; Add the following ingredients to the enzyme-free centrifuge tube in sequence: deionized water, potassium chloride, magnesium sulfate, ammonium sulfate, dNTPs mixture, phenol red, BSA, mannitol, and trehalose, and mix well. Fine-tune the initial pH to 8.6-8.8 using dilute hydrochloric acid / sodium hydroxide; Add SYBR Green I under light-protected conditions and mix well; Finally, add BST DNA polymerase and mix well. After passing quality inspection, the reagents are packaged in the dark to obtain fresh liquid test reagents.

8. The application of the fluorescence / colorimetric dual-mode LAMP detection reagent as described in any one of claims 1-6 in fluorescence and / or colorimetric LAMP nucleic acid detection.

9. A lyophilized formulation, characterized in that, include: The formulation is formed by lyophilizing a mixture of liquid detection reagents and LAMP primers at the working concentration; in The liquid detection reagent is the fluorescence / colorimetric dual-mode LAMP detection reagent as described in any one of claims 1-6.

10. The use of the lyophilized formulation as described in claim 9 in the detection of LAMP nucleic acids using fluorescence and / or colorimetric methods.

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