CN114836522B - New application of ATP in LAMP, LAMP amplification system and kit - Google Patents

Abstract

The application relates to the technical field of isothermal amplification, discloses application of ATP in LAMP for inhibiting nonspecific amplification, and provides a new direction for application of ATP in the technical field of biology. The application also discloses an LAMP amplification system, wherein proper amount of ATP is added in the LAMP amplification system, so that the nonspecific amplification in the amplification process can be obviously inhibited; meanwhile, the reaction time can be shortened, the test cost is saved, and the reaction sensitivity and accuracy are improved. The application also discloses a kit for detecting nucleic acid by using the nucleic acid amplification system, and the kit is less influenced by non-target sequences during detection, high in sensitivity and good in accuracy; the detection period is short, and the target can be detected rapidly within 1 h; the kit is suitable for small and medium-sized units and field detection application.

Description

New application of ATP in LAMP, LAMP amplification system and kit

Technical Field

The application relates to the technical field of isothermal amplification, in particular to a novel application of ATP in LAMP, and also relates to an LAMP amplification system and a kit.

Background

The LAMP (loop-mediated isothermal amplification) technology is characterized in that specific inner primers and outer primers are designed for 6 parts of a target gene, and if necessary, the loop primers can be additionally arranged, so that the target gene can be efficiently amplified in a short time under the constant temperature condition by utilizing a strand displacement reaction, and the LAMP technology is a simple, convenient, rapid, efficient, sensitive, accurate and economic nucleic acid amplification method.

The LAMP technology has very wide application in the field of molecular biology detection, and compared with other existing nucleic acid amplification technologies such as PCR (polymerase chain reaction ) and the like, the LAMP has the unique points: (1) The LAMP technology can realize amplification under isothermal conditions without carrying out the pre-denaturation of a template, reduces the influence of temperature change on an amplification result, reduces the requirement on precision of an experimental instrument, and improves the amplification efficiency; (2) The LAMP technique uses an outer primer, an inner primer and a loop primer, and enhances recognition specificity for a target sequence. The LAMP technology is applied to the fields of pathogenic bacteria, parasites, viruses, diseases, transgenic product detection and the like, and has wider development and application prospects in the fields of clinical diagnosis, environment monitoring, food safety and the like.

However, one of the problems to be solved in the LAMP reaction at present is nonspecific amplification. Since LAMP is performed under isothermal conditions, non-specific amplification not only results in competitive consumption of reagents with specific amplification, but also can result in more non-target amplification products, the presence of which can adversely affect the sensitivity (the smallest detectable template molecule) and specificity (the ability to detect target templates in the presence of competing reactions) of the resulting assay, thereby limiting the limit of detection (LOD) of the assay. Therefore, suppression of non-specific amplification of the LAMP reaction is very important in optimizing analysis of the amplification result.

Therefore, it is of great importance to provide a method capable of simply and efficiently removing non-specific amplification in the LAMP reaction.

Disclosure of Invention

The application aims to: aiming at the current technical problems, one of the purposes of the application is to provide an application of ATP in LAMP to inhibit nonspecific amplification. It is another object of the present application to provide a LAMP-based nucleic acid amplification system in which non-specific amplification during amplification is suppressed by adding an appropriate amount of ATP thereto. The application further aims to provide a kit using the nucleic acid amplification system, which has the advantages of high detection sensitivity, good specificity, simplicity, convenience and rapidness in operation and accurate and reliable results.

In order to achieve the above purpose, the present application provides the following technical solutions:

the application provides an application of ATP in LAMP for inhibiting non-specific amplification, namely ATP can be used as a non-specific amplification inhibitor in LAMP, and the effect is remarkable.

In some embodiments, the concentration of ATP is selected from the group consisting of 0.1 to 3mmol/L, preferably 1.5 to 2.5mmol/L, more preferably 1.6mmol/L, 2mmol/L, 2.5mmol/L. The nucleic acid amplification system is added with a proper amount of ATP, so that nonspecific amplification in the amplification process is inhibited, and meanwhile, the amplification speed is accelerated to a certain extent.

In some embodiments, the amplification template is a DNA template, preferably lambda DNA or GP130, and other types of nucleic acid can be selected by those skilled in the art according to the actual situation.

The application also provides a LAMP amplification system, which comprises: ATP, nucleic acid templates, primers, nucleic acid polymerase, buffers, dNTPs, magnesium sulfate.

In some embodiments, the system further comprises a helicase selected from the group consisting of UvrD enzyme at a concentration of 0.1 ng/. Mu.L to 0.6 ng/. Mu.L, preferably 0.15 ng/. Mu.L to 0.55 ng/. Mu.L, more preferably from 0.16 ng/. Mu.L, 0.32 ng/. Mu.L, 0.4 ng/. Mu.L, 0.48 ng/. Mu.L.

In some embodiments, the nucleic acid template is a DNA template, preferably lambda DNA or GP130, and other types of nucleic acid can be selected by those skilled in the art according to the actual situation.

In some embodiments, the nucleic acid template is lambda DNA, and the primers comprise an upstream inner primer, a downstream inner primer, an upstream outer primer, a downstream outer primer, and a loop primer; the sequence of the upstream inner primer is shown as SEQ NO.1, and the sequence of the downstream inner primer is shown as SEQ NO. 2; the sequence of the upstream outer primer is shown as SEQ NO.3, and the sequence of the downstream outer primer is shown as SEQ NO. 4; the sequence of the loop primer is shown as SEQ NO. 5.

In some embodiments, the nucleic acid template is GP130, and the primers comprise an upstream inner primer, a downstream inner primer, an upstream outer primer, a downstream outer primer, and a loop primer; the sequence of the upstream inner primer is shown as SEQ NO.6, and the sequence of the downstream inner primer is shown as SEQ NO. 7; the sequence of the upstream outer primer is shown as SEQ NO.8, and the sequence of the downstream outer primer is shown as SEQ NO. 9; the sequence of the loop primer is shown as SEQ NO. 10.

In other embodiments, one skilled in the art can select and design the corresponding inner primer, outer primer, and loop primer according to the nucleic acid template to be amplified.

In some embodiments, the nucleic acid polymerase is a DNA polymerase or a functionally active fragment thereof, preferably Bst DNA polymerase or a functionally active fragment thereof. In other embodiments, the DNA polymerase may be selected from Bsm DNA polymerase, phi29 DNA polymerase, equiphi29 DNA polymerase or functional active fragments thereof, and may be selected from other proteins or active fragments thereof capable of performing dNTPs polymerization function in LAMP.

The buffer solution in the system can provide a proper chemical environment for the activity of DNA polymerase and other enzymes in the DNA synthesis process. In some embodiments, the buffer comprises Tris-HCl, KCl, (NH) ₄ ) ₂ SO ₄ 、MgSO ₄ One or more of Triton X-100.

Tris-HCl is the main buffer substance in the buffer solution and is used for regulating the pH value; k provided by KCl ⁺ Can promote the adsorption of the primer; (NH) ₄ ) ₂ SO ₄ NH4 provided ⁺ Can act on weak hydrogen bonds between base pairs of mismatched primer-template complexes to remove their stability, thereby enhancing the specificity of the reaction to some extent.

Magnesium sulfate provides Mg for DNA synthesis ²⁺ ，Mg ²⁺ As a cofactor for DNA polymerase activity, facilitates the polymerization of dNTPs during synthesis. Mg at enzyme active site ²⁺ The phosphodiester bond between the 3' -OH of the primer and the phosphate group of dNTPs can be catalyzed. Also, mg ²⁺ It also stabilizes the negative charge on the phosphate backbone, thereby facilitating the formation of complexes of the primer with the DNA template. Due to Mg ²⁺ Capable of binding dNTPs, primers and DNA templates, and therefore, the general requirement for Mg ²⁺ The concentration is optimized to increase the yield of the amplified product as much as possible and to maintain the amplification specificity thereof. Mg of ²⁺ Too low a concentration reduces polymerase activity, resulting in less or no PCR products; while Mg is ²⁺ Too high a concentration will increase the stability of the primer-template complex, produce non-specific PCR products, and increase replication errors caused by erroneous insertion of dntps. In addition, due to Mg ²⁺ And can react with pyrophosphoric acid ions separated from dNTPs during DNA synthesis to generate magnesium pyrophosphate precipitate, thereby affecting the turbidity of the reaction solution. Thus, the first and second substrates are bonded together,if Mg is ²⁺ Too high concentration may consume too much pyrophosphate ions, reducing the energy required for DNA synthesis, thereby affecting the yield of the target product; the turbidity of the solution caused by the generated pyrophosphatase precipitation may affect the subsequent detection link.

In some embodiments, the composition of the buffer may be adjusted by one skilled in the art according to the specific experimental requirements, and one or more other additives or auxiliary solvents, such as dimethyl sulfoxide (DMSO), glycerol, formamide, bovine Serum Albumin (BSA), polyethylene glycol (PEG), gelatin, betaine (N, N-trimethylglycine), guanidine hydrochloride, etc., may be added thereto. Optionally, triton X-100 may also be replaced with a nonionic detergent such as Tween 20.

In some embodiments, for more stable experimental results, although some MgSO is present in the buffer ₄ It is still necessary to add magnesium sulfate additionally to the system so that the total magnesium sulfate concentration in the system is selected from 4 to 15mmol/L, preferably 6 to 13mmol/L, more preferably 8mmol/L, 10mmol/L, 12mmol/L. Optionally, the magnesium sulfate may also be replaced with magnesium chloride or other magnesium salts.

In some embodiments, the system comprises: ATP 0.1-3 mmol/L, the primer includes: 0.5 to 2.5 mu mol/L of upstream inner primer, 0.5 to 2.5 mu mol/L of downstream inner primer, 0.05 to 0.4 mu mol/L of upstream outer primer, 0.05 to 0.4 mu mol/L of downstream inner primer and 0.4 to 1.2 mu mol/L of loop primer; the nucleic acid polymerase is Bst DNA polymerase 4-10U/. Mu.L, magnesium sulfate 3.9-11 mmol/L, and DNA template 4-4 x106 copies/. Mu.L; the pH of the buffer solution is 7.5-9.4, and the method comprises the following steps: 10-40 mmol/L Tris-HCl, 4-20 mmol/L KCl, (NH) ₄ ) ₂ SO ₄ 4～20mmol/L，MgSO ₄ 0.1-5 mmol/L, and 0.01 w/w-1 w/w% of Triton X-100; dNTPs include: dATP 0.1-3 mmol/L, dGTP 0.1-3 mmol/L, dCTP 0.1-3 mmol/L and dTTP 0.1-3 mmol/L.

In some embodiments, the system comprises: ATP 1.5-2.5 mmol/L, the primer includes: 1.0 to 2.0 mu mol/L of upstream inner primer, 1.0 to 2.0 mu mol/L of downstream inner primer, 0.1 to 0.3 mu mol/L of upstream outer primer, 0.1 to 0.3 mu mol/L of downstream inner primer,0.6 to 1.0 mu mol/L of loop primer; the nucleic acid polymerase is Bst DNA polymerase 5-9U/. Mu.L, magnesium sulfate 5-10 mmol/L, and DNA template 4x10 ¹ ～4x10 ⁵ cobies/. Mu.l; the pH of the buffer solution is 8.0-9.0, and the method comprises the following steps: tris-HCl 15-30 mmol/L, KCl 7-15 mmol/L, (NH) ₄ ) ₂ SO ₄ 7～15mmol/L，MgSO ₄ 1-3 mmol/L, triton X-100.05 w/w% -0.5 w/w%; dNTPs include: dATP 0.5-2 mmol/L, dGTP 0.5-2 mmol/L, dCTP 0.5-2 mmol/L and dTTP 0.5-2 mmol/L.

In some embodiments, the system comprises: ATP 1.6mmol/L, the primer comprises: 1.6 mu mol/L of upstream inner primer, 1.6 mu mol/L of downstream inner primer, 0.2 mu mol/L of upstream outer primer, 0.2 mu mol/L of downstream inner primer and 0.8 mu mol/L of loop primer; the nucleic acid polymerase is Bst DNA polymerase 6-8U/. Mu.L, magnesium sulfate 6mmol/L, DNA template 4x10 ² ～4x10 ⁴ cobies/. Mu.l; the pH of the buffer was 8.8, comprising: tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10mmol/L，MgSO ₄ 2mmol/L, triton X-100.1 w/w%; dNTPs include: dATP 1-1.4 mmol/L, dGTP 1-1.4 mmol/L, dCTP 1-1.4 mmol/L and dTTP 1-1.4 mmol/L.

The final concentration of dNTPs in the reaction system is important for the amplification process of nucleic acids. In the presence of high concentration of Mg ²⁺ In the case of (2), if the concentration of dNTPs is too low, the concentration of dNTPs is too low due to Mg ²⁺ The number of dNTPs which can be introduced during DNA synthesis is reduced by combining with dNTPs, so that the amplification efficiency is reduced; meanwhile, if the concentration of dNTPs exceeds the optimum concentration, the amplification reaction is also inhibited.

In some embodiments, the loop primers may be paired or single-stranded. Loop primers were added to the LAMP amplification system to increase the amplification rate.

The application also provides an LAMP amplification kit comprising the amplification system.

In some embodiments, the kit further comprises a fluorescent dye. The fluorescent dye can be one or more selected from SYBR Green I, miami Yellow, eva Green, nucGreen, gelGreen and Super GelBlue, and can realize fluorescence quantification of nucleic acid.

The application also provides a method for LAMP amplification detection by using the kit, which comprises the following steps:

1) Mixing ATP, a primer, nucleic acid polymerase, a buffer solution, dNTPs, magnesium sulfate and a nucleic acid template in a reaction tube, and adding a fluorescent dye to obtain a mixed solution;

2) And carrying out isothermal amplification reaction on the mixed solution, and detecting.

In some embodiments, the temperature in the isothermal amplification reaction is in the range of 30 to 75 ℃, preferably 50 to 70 ℃, more preferably 60 to 65 ℃.

The beneficial effects are that: compared with the prior art, the application provides the application of ATP in LAMP for inhibiting nonspecific amplification, and provides a new direction for the application of ATP in the biotechnology field.

The application also provides an LAMP amplification system, wherein proper amount of ATP is added in the LAMP amplification system, so that the nonspecific amplification in the amplification process can be obviously inhibited; meanwhile, the reaction time can be shortened, the test cost is saved, and the reaction sensitivity and accuracy are improved.

The application also provides a kit for preparing the amplification system, which has the advantages of little influence by non-target sequences during detection, high sensitivity and good accuracy; the detection period is short, the target can be detected rapidly within 1h, and the kit is suitable for small and medium-sized units and field detection application.

Drawings

FIG. 1 is a diagram of 10 in example 1 ⁶ LAMP amplification plots of lambda DNA for different UvrD enzymes and different ATP concentrations;

FIG. 2 is 10 in example 2 ⁴ LAMP amplification plots for lambda DNA with different UvrD enzymes and different ATP concentrations.

Detailed Description

In order that the application may be readily understood, a detailed description of embodiments of the application will be provided below with reference to specific examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The examples are not intended to identify key or critical elements of the application or to delineate the scope of the application. The materials, reagents or instruments used are commercially available products without identifying the manufacturer.

Specific materials and sources thereof used in embodiments of the present application are provided below. Those skilled in the art will appreciate that these are merely exemplary and are not intended to limit the application, as materials of the same or similar type, model, quality, nature or function as the reagents and instruments described below may be used in the practice of the application.

Unless defined otherwise, 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 in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Terminology

The following are terms used in the following description and claims. However, this term should not be considered separately, and various terms and meanings of the terms in the context of the present application should be fully understood, and the terms should be read in connection with the rest of the specification.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "nucleic acid" is a biological macromolecule, consisting of a plurality of nucleotide molecules. Each nucleotide molecule comprises 1 five carbon sugar, 1 nitrogenous base and several phosphate groups. If the five carbon sugar is deoxyribose, then the nucleotide is deoxyribonucleotide and the polymer is deoxyribonucleic acid (DNA); if the five carbon sugar is ribose, then the nucleotide is a ribonucleotide and the polymer is ribonucleic acid (RNA). "nucleic acids" may be either chain molecules or circular molecules; can be a single-chain molecule or a double-chain molecule; either full-length genome or gene fragment. The "nucleic acid" may be obtained by direct extraction from organisms such as animals, plants, viruses, etc., or may be an artificially synthesized sequence.

The term "ATP" means "Adenine nucleoside triphosphate (abbreviated as "adenosine triphosphate" or "adenosine triphosphate") of formula C ₁₀ H ₁₆ N ₅ O ₁₃ P ₃ The molecular weight is 507.18, and the compound is an unstable high-energy phosphate compound and consists of 1 molecule adenine, 1 molecule ribose and 3 molecules phosphate groups. The elemental composition of ATP is: C. h, O, N, P the molecular formulae A-P to P, in which A represents adenosine, T represents three (the letter T beginning in English triple), P represents a phosphate group, "-" represents a general phosphate bond, "-" represents a special chemical bond, which is called a high-energy phosphate bond (a phosphate bond having an energy of more than 29.32kJ/mol is called a high-energy phosphate bond). 1 "ATP" has 2 high-energy phosphate bonds and 1 common phosphate bond.

The term "dNTP" means "deoxyribonucleotide triphosphate" (dATP) which is a generic term including dATP (adenine deoxynucleotide triphosphate), dGTP (guanine deoxynucleotide triphosphate), dTTP (thymine deoxynucleotide triphosphate), dCTP (cytosine deoxynucleotide triphosphate), dUTP (uracil deoxynucleotide triphosphate) and the like, N means a nitrogen-containing base, and a representative variable means one of A, T, G, C, U and the like. Plays a role as a raw material in the synthesis of biological DNA and various PCR (RT-PCR, real-time PCR) processes.

In the present application, the term "DNA polymerase or functionally active fragment thereof" generally refers to a protein having the function of catalyzing the polymerization of dNTPs molecules to form progeny DNA.

In the present application, the term "Bst DNA polymerase or a functionally active fragment thereof" generally refers to a functional protein having 5'→3' DNA polymerase activity and strong strand displacement activity in Bacillus stearothermophilus DNA polymerase, but lacking 5'→3' exonuclease activity.

In the present application, the term "functionally active fragment" generally refers to a fragment of a larger polypeptide or polynucleotide that retains the same activity or ability as its larger counterpart. The level of activity of the functionally active fragment may be the same as the activity of the larger counterpart, or smaller or larger than it. For example, a functionally active fragment of Bst DNA polymerase may be a polypeptide comprising a smaller number of amino acids than the full length Bst DNA polymerase protein, but still retaining the activity of the full length Bst DNA polymerase.

Experimental materials and instruments

Table 1 materials and instruments involved in the examples

Materials or apparatus	Brand and goods number/model
		ATP	Sigma，A2383-25G
Bst DNA polymerase	Vazyme，P701-02-AA
		Magnesium sulfate	Vazyme，P701-02-AC
10×ThermoPol Buffer	Vazyme，P701-02-AB
		dNTP Mix	Vazyme，P031-02
10×SYBR Green I	Thermo，S7567
		UvrD enzyme	NEB，M1202S
λDNA	NEB，N3011S
		GP130 plasmid	PPL，PPL00004S
Coli bacterium	Vazyme，C505
		Plasmid extraction kit	Vazyme，DC201
qPCR instrument	ABI，QuantStudio 3

Table 2 sequences involved in the examples

Among them, 10×ThermoPol Buffer (Vazyme, P701-02-AB) includes: tris-HCl aqueous solution with initial concentration of 200mmol/L, pH of 8.8, KCl aqueous solution with initial concentration of 100mmol/L, and (NH) with initial concentration of 100mmol/L ₄ ) ₂ SO ₄ Aqueous solution, initial concentration of 20mmol/L MgSO ₄ An aqueous solution, triton X-100 aqueous solution with an initial mass fraction of 1%.

dNTP Mix (Vazyme, P031-02) includes: dATP, dCTP, dTTP, dGTP at a concentration of 10mmol/L each.

Example 1

LAMP assay 10 Using the above reagents ⁶ A copies lambda DNA comprising the steps of:

1. setting of experimental groups:

in this example, an experimental group and a comparative test group were simultaneously set, and each system was 25. Mu.l. ATP was added to the test group and the same amount of ddH was used in the control group ₂ O replaces ATP, wherein templates are 10 ⁶ cobies lambda DNA. The experimental and comparative test groups were each provided with a negative control, in which the template was replaced with an equal amount of water, and the remaining conditions were identical to those of the experimental group (Table 3).

Table 3 list of experimental conditions of example 1

2. Loop-mediated isothermal amplification of lambda DNA:

(1) Preparation of LAMP reaction System (25. Mu.l):

positive reaction systems of the experimental and comparative test groups were prepared according to Table 4, each of which was placed in a single reaction tube. All positive 1 to positive 6 shown in Table 4 were negative controls, and only 5. Mu.l of water was used instead of lambda DNA template.

Table 4 10 ⁶ cobies lambda DNA positive reaction system

(2) Loop-mediated isothermal amplification: the reaction tube with the reaction system was placed in a qPCR apparatus, and reacted at a constant temperature of 63℃for 1 hour, and after the completion of the reaction, the results were shown in FIG. 1.

3. Reaction results:

as can be seen from FIG. 1, under the reaction conditions of example 1, the positive amplification rate was slower and the negative amplification rate was faster when the system did not contain UvrD enzyme and ATP. When the system does not contain UvrD enzyme and 1.6mmol/LATP is added, the positive amplification speed is increased, and negative amplification does not occur. When UvrD enzyme was added to a 1.6mmol/LATP containing system, no amplification occurred in the negative; when 4-8 ng of UvrD enzyme is added, compared with the case of not adding UvrD enzyme, the amplification speed is unchanged, and the positive platform is slightly reduced; when UvrD increases to 10ng, the positive plateau drops significantly; when UvrD was increased to 12ng, positivity was not amplified.

Example 2

LAMP assay 10 Using the above reagents ⁴ A copies lambda DNA comprising the steps of:

1. setting of experimental groups:

in this example, an experimental group and a comparative test group were simultaneously set, and each system was 25. Mu.l. ATP is added into the experimental group, and the comparative detection group uses the same amount of water to replace ATP, wherein the templates are 10 ⁴ cobies lambda DNA. The experimental group and the contrast detection group are respectively required to be provided with negative control, and the negative control is formed by equal ddH ₂ O replaces the template, the remaining conditions were identical to the experimental group (table 5).

Table 5 list of experimental conditions of example 2

2. Loop-mediated isothermal amplification of lambda DNA:

(1) Configuration of LAMP reaction System (25. Mu.l):

positive reaction systems of the experimental and comparative test groups were prepared according to Table 6, each of which was placed in a single reaction tube. All positive 1 to positive 5 shown in Table 6 were negative controls, and only 5. Mu.l of water was used instead of lambda DNA template.

TABLE 6 10 ⁴ cobies lambda DNA positive reaction system

(2) Loop-mediated isothermal amplification: the reaction tube with the reaction system was placed in a qPCR apparatus, and reacted at a constant temperature of 63℃for 1 hour, and after the completion of the reaction, the results were shown in FIG. 2.

3. Reaction results:

as can be seen from FIG. 2, under the reaction conditions of example 2, the positive amplification rate was slower and the negative amplification rate was faster when the system did not contain UvrD enzyme and ATP. When the system does not contain UvrD enzyme and 1.6mmol/LATP is added, the positive amplification speed is increased, and negative amplification does not occur. When UvrD enzyme was added to a 1.6mmol/LATP containing system, no amplification occurred in the negative; when 4ng of UvrD enzyme was added, the amplification rate was unchanged compared to the case without UvrD enzyme; when UvrD increases to 8ng, the positive platform drops significantly and the negative does not amplify; when UvrD was increased to 10ng, no amplification occurred in the positive.

Example 3

In this example, the reagent was used to screen for optimal ATP and MgSO4 concentrations suitable for LAMP without addition of UvrD enzyme, comprising the steps of:

extraction of GP130:

transferring the GP130 plasmid directly into escherichia coli, culturing under Kan resistance, and extracting GP130 by using a plasmid extraction kit after culturing.

2. Setting of experimental groups:

the present embodiment sets three sets of variables: (1) templates of different kinds and numbers: 10 ⁴ copies GP130、10 ⁶ copies GP130、10 ⁴ cobies lambda DNA and 10 ⁶ cobies lambda DNA; (2) ATP at different concentrations: 0mmol/L, 1.6mmol/L, 2mmol/L, 2.5mmol/L; (3) MgSO at different total concentrations ₄ :8mmol/L, 10mmol/L, 12mmol/L. The crossover experiments were set up using the three variables described above, resulting in 64 positive experimental groups (e.g., tables 7, 8) of 25 μl each.

The positive test groups all need to be provided with negative control, and the negative control is composed of equal ddH ₂ O replaces the template, and the rest conditions are consistent with those of the positive experiment group, so that 64 negative control groups (NTCs) are obtained.

3. Loop-mediated isothermal amplification:

(1) Configuration of LAMP reaction System (25. Mu.l):

the reaction system was formulated as in table 4, except that: no UvrD enzyme was added, and an appropriate amount of MgSO was added ₄ The ATP and the template are required to meet the concentration in the corresponding system of the table 7 or the table 8, the primers corresponding to the GP130 are respectively and correspondingly replaced by SEQ NO. 1-5 to SEQ NO. 6-10, and ddH is used for the volume shortage ₂ And O is complemented.

All positive controls described above were set up with negative controls, with only 5 μl water being used instead of the corresponding template.

(2) Loop-mediated isothermal amplification: the reaction tube with the reaction system was placed in a qPCR apparatus and reacted at a constant temperature of 63℃for 1 hour.

(3) Calculation of Ct value: after completion of the reaction, ct values of the reaction were calculated, and the results are shown in Table 7 and Table 8.

TABLE 7 ATP/MgSO with GP130 as template ₄ Concentration cross screening experiment

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TABLE 8 ATP/MgSO4 concentration Cross screening experiments with lambda DNA as template

Finally, it should be noted that the foregoing embodiments only show a part of the results for illustrating the technical solution of the present application, not all the results, but not the limitation of the scope of the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the present application.

Sequence listing

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Claims (19)

1. The use of ATP for inhibiting non-specific amplification in LAMP, wherein the ATP concentration in the LAMP amplification system is 1.6mmol/L.
2. 2. The use according to claim 1, characterized in that the LAMP amplification system further comprises 4-15 mmol/L magnesium sulfate.
3. 3. The use according to claim 1, characterized in that the LAMP amplification system consists of ATP, nucleic acid templates, primers, nucleic acid polymerase, buffers, dNTPs, magnesium sulfate.
4. 4. The use according to claim 1, wherein the nucleic acid template is a DNA template selected from lambda DNA or GP130 plasmid, wherein the GP130 plasmid is purchased from PPL under the accession number PPL00004S.
5. 5. A LAMP amplification system, characterized in that: the system consists of ATP, a nucleic acid template, a primer, nucleic acid polymerase, a buffer solution, dNTPs and magnesium sulfate;

   the nucleic acid template is lambda DNA, and the primer comprises an upstream inner primer, a downstream inner primer, an upstream outer primer, a downstream outer primer and a loop primer; the sequence of the upstream inner primer is shown as SEQ NO.1, and the sequence of the downstream inner primer is shown as SEQ NO. 2; the sequence of the upstream outer primer is shown as SEQ NO.3, and the sequence of the downstream outer primer is shown as SEQ NO. 4; the sequence of the loop primer is shown as SEQ NO. 5;

   the ATP is 1.6mmol/L, and the primer comprises: 0.5-2.5 mu mol/L of upstream inner primer, 0.5-2.5 mu mol/L of downstream inner primer, 0.05-0.4 mu mol/L of upstream outer primer, 0.05-0.4 mu mol/L of downstream outer primer and 0.4-1.2 mu mol/L of loop primer; 4-10U/. Mu.L of the nucleic acid polymerase, 3.9-11 mmol/L of the magnesium sulfate and 4-4 x10 of the lambda DNA ⁶ cobies/. Mu.l; the pH of the buffer solution is 7.5-9.4, and the buffer solution comprises: 10-40 mmol/L Tris-HCl, 4-20 mmol/L KCl, (NH) ₄ ) ₂ SO ₄ 4～20mmol/L，MgSO ₄ 0.1-5 mmol/L, triton X-1000.01w/w% -1 w/w%; the dNTPs include: dATP 0.1-3 mmol/L, dGTP 0.1-3 mmol/L, dCTP 0.1-3 mmol/L and dTTP 0.1-3 mmol/L.
6. 6. The amplification system of claim 5, wherein the ATP is at 1.6mmol/L and the primer comprises: 1.0 to 2.0 mu mol/L of upstream inner primer, 1.0 to 2.0 mu mol/L of downstream inner primer, 0.1 to 0.3 mu mol/L of upstream outer primer, 0.1 to 0.3 mu mol/L of downstream outer primer and 0.6 to 1.0 mu mol/L of loop primer; 5-9U/. Mu.L of the nucleic acid polymerase, 5-10 mmol/L of the magnesium sulfate and 4x10 of the lambda DNA ¹ ～4x10 ⁵ cobies/. Mu.l; the pH of the buffer solution is 8.0-9.0, and the buffer solution comprises: tris-HCl 15-30 mmol/L, KCl 7-15 mmol/L, (NH) ₄ ) ₂ SO ₄ 7～15mmol/L，MgSO ₄ 1-3 mmol/L, triton X-1000.05w/w% -0.5 w/w%; the dNTPs include: dATP 0.5-2 mmol/L, dGTP 0.5-2 mmol/L, dCTP 0.5-2 mmol/L and dTTP 0.5-2 mmol/L.
7. 7. The amplification system of claim 5, wherein: by a means ofThe ATP is 1.6mmol/L, and the primer comprises: 1.6 mu mol/L of upstream inner primer, 1.6 mu mol/L of downstream inner primer, 0.2 mu mol/L of upstream outer primer, 0.2 mu mol/L of downstream outer primer and 0.8 mu mol/L of loop primer; 6-8U/. Mu.L of the nucleic acid polymerase, 6mmol/L of the magnesium sulfate, and 4x10 of the lambda DNA ² ～4x10 ⁴ cobies/. Mu.l; the pH of the buffer was 8.8, comprising: tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10 mmol/L，MgSO ₄ 2mmol/L, triton X-1000.1w/w%; the dNTPs include: dATP 1-1.4 mmol/L, dGTP 1-1.4 mmol/L, dCTP 1-1.4 mmol/L and dTTP 1-1.4 mmol/L.
8. 8. The amplification system of claim 5, wherein: the ATP is 1.6mmol/L, and the primer comprises: 1.6 mu mol/L of upstream inner primer, 1.6 mu mol/L of downstream inner primer, 0.2 mu mol/L of upstream outer primer, 0.2 mu mol/L of downstream outer primer and 0.8 mu mol/L of loop primer; the nucleic acid polymerase 8U/. Mu.L, the magnesium sulfate 6mmol/L, the lambda DNA 10 ⁶ cobies/. Mu.l; the pH of the buffer was 8.8, comprising: tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10 mmol/L，MgSO ₄ 2mmol/L, triton X-1000.1w/w%; the dNTPs include: dATP 1.4mmol/L, dGTP 1.4mmol/L, dCTP 1.4mmol/L, dTTP1.4 mmol/L; the system further comprises a fluorescent dye 10 x SYBR Green I and a helicase, wherein the volume percentage of the fluorescent dye 10 x SYBR Green I is 2%, the helicase is selected from UvrD enzyme, and the concentration of the UvrD enzyme is not more than 0.4 ng/. Mu.L.
9. 9. The amplification system of claim 5, wherein: the ATP is 1.6mmol/L, and the primer comprises: 1.6 mu mol/L of upstream inner primer, 1.6 mu mol/L of downstream inner primer, 0.2 mu mol/L of upstream outer primer, 0.2 mu mol/L of downstream outer primer and 0.8 mu mol/L of loop primer; the nucleic acid polymerase 8U/. Mu.L, the magnesium sulfate 6mmol/L, the lambda DNA 10 ⁴ cobies/. Mu.l; the pH of the buffer was 8.8, comprising: tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10 mmol/L，MgSO ₄ 2 mmol/L，Triton X-1000.1w/w％；The dNTPs include: dATP 1.4mmol/L, dGTP 1.4mmol/L, dCTP 1.4mmol/L, dTTP1.4 mmol/L; the system further comprises a fluorescent dye 10 x SYBR Green I and a helicase, wherein the volume percentage of the fluorescent dye 10 x SYBR Green I is 2%, the helicase is selected from UvrD enzyme, and the concentration of the UvrD enzyme is not more than 0.32 ng/. Mu.L.
10. 10. A LAMP amplification system, characterized in that: the system consists of ATP, a nucleic acid template, a primer, nucleic acid polymerase, a buffer solution, dNTPs and magnesium sulfate;

    the nucleic acid template is GP130 plasmid, the GP130 plasmid is purchased from PPL, the product number is PPL00004S, and the primers comprise an upstream inner primer, a downstream inner primer, an upstream outer primer, a downstream outer primer and a loop primer; the sequence of the upstream inner primer is shown as SEQ NO.6, and the sequence of the downstream inner primer is shown as SEQ NO. 7; the sequence of the upstream outer primer is shown as SEQ NO.8, and the sequence of the downstream outer primer is shown as SEQ NO. 9; the sequence of the loop primer is shown as SEQ NO. 10;

    the ATP is 1.6-2.5 mmol/L, and the primer comprises: 0.5-2.5 mu mol/L of upstream inner primer, 0.5-2.5 mu mol/L of downstream inner primer, 0.05-0.4 mu mol/L of upstream outer primer, 0.05-0.4 mu mol/L of downstream outer primer and 0.4-1.2 mu mol/L of loop primer; 4-10U/mu L of the nucleic acid polymerase, 3.9-11 mmol/L of magnesium sulfate and 4-4 x10 of the nucleic acid template ⁶ cobies/. Mu.l; the pH of the buffer solution is 7.5-9.4, and the buffer solution comprises: 10-40 mmol/L Tris-HCl, 4-20 mmol/L KCl, (NH) ₄ ) ₂ SO ₄ 4～20mmol/L，MgSO ₄ 0.1-5 mmol/L, triton X-1000.01w/w% -1 w/w%; the dNTPs include: dATP 0.1-3 mmol/L, dGTP 0.1-3 mmol/L, dCTP 0.1-3 mmol/L and dTTP 0.1-3 mmol/L.
11. 11. The amplification system of claim 10, wherein: the primer comprises: 1.0 to 2.0 mu mol/L of upstream inner primer, 1.0 to 2.0 mu mol/L of downstream inner primer, 0.1 to 0.3 mu mol/L of upstream outer primer, 0.1 to 0.3 mu mol/L of downstream outer primer and 0.6 to 1.0 mu mol/L of loop primer; 5-9U/mu L of the nucleic acid polymerase and 5-10 magnesium sulfatemmol/L, the nucleic acid template is 4x10 ¹ ～4x10 ⁵ cobies/. Mu.l; the pH of the buffer solution is 8.0-9.0, and the buffer solution comprises: tris-HCl 15-30 mmol/L, KCl 7-15 mmol/L, (NH) ₄ ) ₂ SO ₄ 7～15mmol/L，MgSO ₄ 1-3 mmol/L, triton X-1000.05w/w% -0.5 w/w%; the dNTPs include: dATP 0.5-2 mmol/L, dGTP 0.5-2 mmol/L, dCTP 0.5-2 mmol/L and dTTP 0.5-2 mmol/L.
12. 12. The amplification system of claim 10, wherein: the ATP is 1.6mmol/L, and the primer comprises: 1.6 mu mol/L of upstream inner primer, 1.6 mu mol/L of downstream inner primer, 0.2 mu mol/L of upstream outer primer, 0.2 mu mol/L of downstream outer primer and 0.8 mu mol/L of loop primer; 6-8U/. Mu.L of the nucleic acid polymerase, 6mmol/L of the magnesium sulfate and 4x10 of the nucleic acid template ² ～4x10 ⁴ cobies/. Mu.l; the pH of the buffer was 8.8, comprising: tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10 mmol/L，MgSO ₄ 2mmol/L, triton X-1000.1w/w%; the dNTPs include: dATP 1-1.4 mmol/L, dGTP 1-1.4 mmol/L, dCTP 1-1.4 mmol/L and dTTP 1-1.4 mmol/L.
13. 13. The amplification system of claim 5 or 10, wherein: the nucleic acid polymerase is a DNA polymerase or a functionally active fragment thereof.
14. 14. The amplification system of claim 5 or 10, wherein: the nucleic acid polymerase is Bst DNA polymerase or a functionally active fragment thereof.
15. 15. A LAMP amplification kit comprising the amplification system of any one of claims 5 to 14.
16. 16. A method for LAMP amplification detection using the kit of claim 15, comprising the steps of:

    1) Mixing ATP, a primer, nucleic acid polymerase, a buffer solution, dNTPs, magnesium sulfate and a nucleic acid template in a reaction tube, and adding a fluorescent dye to obtain a mixed solution;

    2) And carrying out isothermal amplification reaction on the mixed solution, and detecting.
17. 17. The method according to claim 16, wherein: the temperature range of the isothermal amplification reaction is 30-75 ℃.
18. 18. The method according to claim 16, wherein: the temperature range of the isothermal amplification reaction is 50-70 ℃.
19. 19. The method according to claim 16, wherein: the temperature range of the isothermal amplification reaction is 60-65 ℃.