CN114836522A

CN114836522A - Novel application of ATP in LAMP, LAMP amplification system and kit

Info

Publication number: CN114836522A
Application number: CN202210309511.6A
Authority: CN
Inventors: 张力军; 赵敬发; 时洁
Original assignee: Nanjing Nuoweizan Animal Health Co ltd; Nanjing Novozan Biotechnology Co ltd
Current assignee: Nanjing Nuoweizan Animal Health Co ltd; Nanjing Novozan Biotechnology Co ltd
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-08-02
Anticipated expiration: 2042-03-28
Also published as: CN114836522B

Abstract

The invention relates to the technical field of isothermal amplification, discloses application of ATP in inhibiting nonspecific amplification in LAMP, and provides a new direction for application of ATP in the technical field of biology. The invention also discloses an LAMP amplification system, wherein a proper amount of ATP is added, so that non-specific 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 invention also discloses a kit for detecting nucleic acid by using the nucleic acid amplification system, which is little affected by non-target sequences during detection, high in sensitivity and good in accuracy; the detection period is short, and the target can be quickly detected within 1 h; the kit is suitable for small and medium-sized units and field detection.

Description

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

Technical Field

The invention relates to the technical field of isothermal amplification, in particular to a new 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 specific outer primers are designed for 6 parts of a target gene, if necessary, a loop primer can be added, and the target gene is efficiently amplified in a short time under a constant temperature condition by using a strand displacement reaction, so that the LAMP (loop-mediated isothermal amplification) technology is a simple, convenient, rapid, efficient, sensitive, accurate and economic nucleic acid amplification method.

The LAMP technology is widely applied in the field of molecular biology detection, and compared with other existing nucleic acid amplification technologies such as PCR (polymerase chain reaction), LAMP has the unique characteristics that: (1) the LAMP technology can realize amplification under the isothermal condition without carrying out pre-denaturation of a template, thereby reducing the influence of temperature change on an amplification result, lowering the requirement on the precision of an experimental instrument and improving the amplification efficiency; (2) the LAMP technology uses an outer primer, an inner primer and a loop primer, and the recognition specificity of a target sequence is enhanced. LAMP technology is applied to the fields of detection of pathogenic bacteria, parasites, viruses, diseases, transgenic products and the like, and has wider development and application prospects in the fields of clinical diagnosis, environmental monitoring, food source safety and the like.

However, one of the problems to be solved in the LAMP reaction is non-specific amplification. Since LAMP is performed under isothermal conditions, non-specific amplification not only competitively consumes reagents with specific amplification, but also produces more non-target amplification products whose presence adversely affects the sensitivity (the fewest template molecules that can be detected) and specificity (the ability to detect the target template in the presence of competing reactions) of the resulting assay, thereby limiting the limit of detection (LOD) of the assay. Therefore, inhibition of non-specific amplification by LAMP reaction is very important in optimizing the analysis of amplification results.

Therefore, it is very important to provide a method for removing non-specific amplification in LAMP reaction simply and efficiently.

Disclosure of Invention

The purpose of the invention is as follows: in view of the current technical problems, one of the objects of the present invention is to provide the use of ATP for the inhibition of non-specific amplification in LAMP. It is another object of the present invention to provide a LAMP-based nucleic acid amplification system in which nonspecific amplification during amplification is suppressed by adding an appropriate amount of ATP thereto. The invention also aims to provide a kit using the nucleic acid amplification system, which has the advantages of high detection sensitivity, good specificity, simple and quick operation and accurate and reliable result.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides the application of ATP in inhibiting nonspecific amplification in LAMP, namely ATP can be used as a nonspecific amplification inhibitor in LAMP, and the effect is obvious.

In some embodiments, the concentration of ATP is selected from 0.1-3 mmol/L, preferably 1.5-2.5 mmol/L, more preferably 1.6mmol/L, 2mmol/L, 2.5 mmol/L. The proper amount of ATP is added in the nucleic acid amplification system, so that nonspecific amplification in the amplification process is inhibited, and 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 one skilled in the art can select other types of nucleic acid species as appropriate.

The invention also provides an LAMP amplification system, which comprises: ATP, nucleic acid template, primer, nucleic acid polymerase, buffer solution, dNTPs and magnesium sulfate.

In some embodiments, the system further comprises a helicase that selects for the 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 one skilled in the art can select other types of nucleic acid species 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 corresponding inner primers, outer primers, and loop primers 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 functionally active fragments thereof, or from other proteins or active fragments thereof that function to polymerize dNTPs in LAMP.

The buffer in the system can provide a suitable chemical environment for the DNA polymerase and other enzyme activities in the DNA synthesis process. In some embodiments, the buffer comprises Tris-HCl, KCl, (NH) ₄ ) ₂ SO ₄ 、MgSO ₄ And Triton X-100.

Tris-HCl is a main buffering substance in the buffer solution and is used for adjusting the pH value; k provided by KCl ⁺ The adsorption of the primer can be promoted; (NH) ₄ ) ₂ SO ₄ NH4 supplied ⁺ Can act on weak hydrogen bonds between base pairs of a mismatched primer-template complex to remove the stability of the base pairs, thereby enhancing the specificity of the reaction to a certain extent.

Magnesium sulfate can provide Mg for DNA synthesis process ²⁺ ，Mg ²⁺ As a cofactor for DNA polymerase activity, it contributes to the polymerization of dNTPs during synthesis. Mg at the enzyme active site ²⁺ Can catalyze the formation of phosphodiester bonds between the 3' -OH of the primer and the phosphate groups of the dNTPs. And, Mg ²⁺ It also stabilizes the negative charge on the phosphate backbone, thereby facilitating the formation of a complex between the primer and the DNA template. Due to Mg ²⁺ Capable of binding dNTPs, primers and DNA templates, and therefore, the need for Mg is generally present ²⁺ The concentration is optimized to maximize the yield of the amplification product and maintain the amplification specificity. Mg (magnesium) ²⁺ Too low a concentration will decrease polymerase activity, resulting in fewer or no PCR products; and Mg ²⁺ Too high a concentration increases the stability of the primer-template complex, produces non-specific PCR products, and increases replication errors due to dNTP misincorporations. Furthermore, due to Mg ²⁺ It can also react with pyrophosphate ions precipitated from dNTPs during DNA synthesis to form magnesium pyrophosphate precipitates, thereby affecting the turbidity of the reaction solution. Therefore, if Mg ²⁺ Too high concentration may consume too much pyrophosphate ions, so that the energy required for DNA synthesis is reduced, thereby affecting the yield of the target product; the turbidity degree of the solution caused by the generated pyrophosphatase precipitate may influence the subsequent detection process.

In some embodiments, the buffer composition 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, and the like, may be added thereto. Alternatively, Triton X-100 can also be replaced with a non-ionic detergent such as Tween 20.

In some technical schemes, for the purpose of more stable experiment effect, although partial MgSO is already existed in the buffer solution ₄ However, magnesium sulfate is still added to the system to make the total magnesium sulfate concentration in the system selected from 4-15 mmol/L, preferably 6-13 mmol/L, more preferably 8mmol/L, 10mmol/L, 12 mmol/L. Alternatively, the magnesium sulfate may also be replaced with magnesium chloride or other magnesium salts.

In some embodiments, the system comprises: 0.1-3 mmol/L of ATP, and primers comprise: upstream inner primer 0.5-2.5 mu mol/L, downstream inner primer 0.5-2.5 mu mol/L, upstream outer primer 0.05-0.4 mu mol/L, downstream inner primer0.05-0.4 mu mol/L of the compound, and 0.4-1.2 mu mol/L of the loop primer; the nucleic acid polymerase is Bst DNA polymerase 4-10U/mu L, magnesium sulfate 3.9-11 mmol/L, DNA template 4-4 x106 copies/mu L; the pH value of the buffer solution is 7.5-9.4, and the buffer solution comprises: Tris-HCl 10-40 mmol/L, KCl 4-20 mmol/L, (NH) ₄ ) ₂ SO ₄ 4～20mmol/L，MgSO ₄ 0.1-5 mmol/L, Triton X-1000.01 w/w% -1 w/w%; the dNTPs include: 0.1-3 mmol/L of dATP, 0.1-3 mmol/L of dGTP, 0.1-3 mmol/L of dCTP and 0.1-3 mmol/L of dTTP.

In some embodiments, the system comprises: ATP is 1.5-2.5 mmol/L, and the primers comprise: 1.0-2.0 mu mol/L of upstream inner primers, 1.0-2.0 mu mol/L of downstream inner primers, 0.1-0.3 mu mol/L of upstream outer primers, 0.1-0.3 mu mol/L of downstream inner primers and 0.6-1.0 mu mol/L of loop primers; the nucleic acid polymerase is Bst DNA polymerase 5-9U/microliter, magnesium sulfate 5-10 mmol/microliter, DNA template 4x10 ¹ ～4x10 ⁵ copies/. 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-1000.05 w/w% -0.5 w/w%; the dNTPs include: 0.5-2 mmol/L of dATP, 0.5-2 mmol/L of dGTP, 0.5-2 mmol/L of dCTP and 0.5-2 mmol/L of dTTP.

In some embodiments, the system comprises: ATP 1.6mmol/L, and the primers comprise: upstream inner primer 1.6 mu mol/L, downstream inner primer 1.6 mu mol/L, upstream outer primer 0.2 mu mol/L, downstream inner primer 0.2 mu mol/L, and loop primer 0.8 mu mol/L; the nucleic acid polymerase is Bst DNA polymerase 6-8U/microliter, magnesium sulfate 6 mmol/liter, DNA template 4x10 ² ～4x10 ⁴ copies/. mu.l; the pH of the buffer was 8.8, including: Tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10mmol/L，MgSO ₄ 2mmol/L, Triton X-1000.1 w/w%; the dNTPs include: 1-1.4 mmol/L of dATP, 1-1.4 mmol/L of dGTP, 1-1.4 mmol/L of dCTP and 1-1.4 mmol/L of dTTP.

The final concentration of dNTPs in the reaction system is important for the amplification process of nucleic acid. In the presence of high concentration of Mg ²⁺ In the case of (2), if the concentration of dNTPs is too low, Mg is included ²⁺ Junctions with dNTPsIn addition, the quantity of dNTPs which can be introduced during DNA synthesis is reduced, and the amplification efficiency is reduced; also, if the concentration of dNTPs exceeds the optimum concentration, the amplification reaction is also inhibited.

In some embodiments, the loop primers can be paired or single. The loop primer is added to the LAMP amplification system in order to increase the amplification speed.

The invention also provides a LAMP amplification kit comprising the amplification system.

In some embodiments, the kit further comprises a fluorescent dye. The fluorescent dye can be selected from one or more of SYBR Green I, Miami Yellow, Eva Green, NucGreen, GelGreen and Super GelBlue, and can realize the fluorescent quantification of nucleic acid.

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

1) mixing ATP, primers, nucleic acid polymerase, 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 for detection.

In some technical schemes, the temperature range of the isothermal amplification reaction is 30-75 ℃, preferably 50-70 ℃, and more preferably 60-65 ℃.

Has the advantages that: compared with the prior art, the invention provides the application of ATP in the LAMP for inhibiting nonspecific amplification, and provides a new direction for the application of ATP in the field of biotechnology.

The invention also provides an LAMP amplification system, wherein a proper amount of ATP is added, so that non-specific 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 invention also provides a kit for preparing the amplification system, which is little affected by non-target sequences during detection, high in sensitivity and good in accuracy; the detection period is short, the target can be quickly detected within 1h, and the kit is suitable for small and medium-sized units and field detection application.

Drawings

FIG. 1 shows 10 in example 1 ⁶ LAMP amplification curve graphs of different UvrD enzymes and different ATP concentrations of lambda DNA;

FIG. 2 shows example 2, 10 ⁴ LAMP amplification profiles of lambda DNA at different UvrD enzymes and different ATP concentrations.

Detailed Description

In order that the invention may be readily understood, embodiments of the invention are described in detail below with reference to specific examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The specific techniques or conditions are not indicated in the examples, and are performed according to the conventional techniques or conditions in the field or according to the product specification. The materials, reagents or instruments used are not indicated by manufacturers, and are all commercially available products.

The following provides specific materials and sources thereof used in embodiments of the present invention. It will be understood by those skilled in the art that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the reagents and instruments described below may be used in the practice of the invention.

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Term(s) for

The following are terms used in the following specification and claims. However, this term should not be considered separately, and for a complete understanding of the various terms and their meanings in the context of the present invention, the terms should be read in conjunction with the remainder of this 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 includes 1 five-carbon sugar, 1 nitrogenous base, and several phosphate groups. If the five-carbon sugar is deoxyribose, the nucleotide is deoxyribonucleotide, and the polymer thereof is deoxyribonucleic acid (DNA); if the five-carbon sugar is ribose, the nucleotide is a ribonucleotide and the polymer is ribonucleic acid (RNA). The "nucleic acid" may be a chain molecule or a cyclic molecule; can be single-chain molecule or double-chain molecule; the gene may be a full-length genome or a gene fragment. The "nucleic acid" may be obtained by direct extraction from animals, plants, viruses, and other organisms, or may be a sequence artificially synthesized.

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

The term "dNTP" means "deoxyribonucleotide triphosphate" (deoxyribotide triphosphate), 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), etc., and N means a nitrogenous base, which represents one of the variables A, T, G, C, U, etc. Plays a role as a raw material in the biological DNA synthesis 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 that has 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 of Bacillus stearothermophilus DNA polymerase having 5 '→ 3' DNA polymerase activity and strong strand displacement activity, 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 a functionally active fragment may be the same as, less than, or greater than the activity of the larger counterpart. For example, a functionally active fragment of Bst DNA polymerase can be a polypeptide that consists of fewer amino acids than the full-length Bst DNA polymerase protein, but still retains the activity of the full-length Bst DNA polymerase.

Experimental materials and instruments

Table 1 materials and instruments referred to in the examples

Materials or instruments	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
Escherichia coli	Vazyme，C505
		Plasmid extraction kit	Vazyme，DC201
qPCR instrument	ABI，QuantStudio 3

Sequences referred to in the example of Table 2

Among them, 10X 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, (N) with initial concentration of 100mmol/LH ₄ ) ₂ SO ₄ Aqueous solution, 20mmol/L initial MgSO ₄ Aqueous solution, Triton X-100 aqueous solution with the initial mass fraction of 1%.

Among dNTP Mix (Vazyme, P031-02) are: dATP, dCTP, dTTP and dGTP were added at concentrations of 10mmol/L, respectively.

Example 1

Detection by LAMP 10 Using the above-described reagent ⁶ copies lambda DNA comprising the steps of:

1. setting of experimental group:

in this example, an experimental group and a comparative detection group were set simultaneously, and each system was 25. mu.l. ATP is added in the experimental group, and equal amount of ddH is used in the contrast detection group ₂ O instead of ATP, wherein the templates are all 10 ⁶ copies lambda DNA. The experimental group and the comparative detection group are both required to be provided with negative control, the template is replaced by equal amount of water in the negative control, and the rest conditions are consistent with those of the experimental group (Table 3).

Table 3 list of experimental conditions for 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 group and the comparative detection group were prepared according to Table 4, and each system was placed in a single reaction tube. In Table 4, the positive 1 to the positive 6 were all negative controls, and 5. mu.l of water was used instead of the lambda DNA template.

Table 410 ⁶ copies lambda DNA positive reaction system

(2) Loop-mediated isothermal amplification: the reaction tube with the reaction system is placed in a qPCR instrument and reacted for 1h at a constant temperature of 63 ℃, and after the reaction is finished, the obtained result is shown in figure 1.

3. And (3) reaction results:

as is clear from FIG. 1, under the reaction conditions of example 1, when UvrD enzyme and ATP were not contained in the system, the positive amplification rate was slow and the negative amplification rate was fast. When the system does not contain UvrD enzyme and 1.6mmol/LATP is added, the positive amplification speed becomes fast, and the negative amplification does not occur. When UvrD enzyme is added into a system containing 1.6mmol/LATP, negative amplification does not occur; when 4-8 ng of UvrD enzyme is added, compared with the condition that UvrD enzyme is not added, the amplification speed is unchanged, and a positive platform is slightly reduced; when UvrD increased to 10ng, the positive plateau dropped significantly; when UvrD increased to 12ng, positives were not amplified.

Example 2

Detection by LAMP 10 Using the above-described reagents ⁴ copies lambda DNA comprising the steps of:

1. setting of experimental group:

in this example, an experimental group and a comparative detection group were set simultaneously, and each system was 25. mu.l. ATP is added into the experimental group, the ATP is replaced by the same amount of water in the contrast detection group, and the templates are all 10 ⁴ copies lambda DNA. The experimental group and the comparison detection group are both required to be provided with negative control, and the negative control consists of equal ddH ₂ O replaced the template and the remaining conditions were consistent with the experimental groups (Table 5).

Table 5 list of experimental conditions for example 2

2. Loop-mediated isothermal amplification of lambda DNA:

(1) configuration of LAMP reaction System (25. mu.l):

positive reaction systems for the experimental group and the comparative detection group were prepared according to Table 6, and each system was placed in a single reaction tube. In Table 6, the positive 1 to the positive 5 were each provided with a negative control, and 5. mu.l of water was used instead of the lambda DNA template.

Table 610 ⁴ copies 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 63 ℃ for 1h, and the results after the reaction were completed are shown in FIG. 2.

3. And (3) reaction results:

as is clear from FIG. 2, under the reaction conditions of example 2, when UvrD enzyme and ATP were not contained in the system, the positive amplification rate was slow, and the negative amplification rate was fast. When the system does not contain UvrD enzyme and 1.6mmol/LATP is added, the positive amplification speed becomes fast, and the negative amplification does not occur. When UvrD enzyme is added into a system containing 1.6mmol/LATP, negative amplification does not occur; when 4ng of UvrD enzyme is added, the amplification speed is unchanged compared with that without the UvrD enzyme; when UvrD is increased to 8ng, the positive platform is obviously reduced, and the negative platform is not amplified; when UvrD was increased to 10ng, no amplification occurred for positives.

Example 3

This example used the above reagents to screen for optimal ATP and MgSO4 concentrations for LAMP without the addition of UvrD enzyme, including the following steps:

extraction of GP 130:

the GP130 plasmid is directly transferred into escherichia coli, the escherichia coli is cultured under Kan resistance, and the GP130 is extracted by a plasmid extraction kit after the culture is finished.

2. Setting of experimental group:

this embodiment sets three sets of variables: (1) different kinds and numbers of templates: 10 ⁴ copies GP130、10 ⁶ copies GP130、10 ⁴ copies Lambda DNA and 10 ⁶ copies lambda DNA; (2) ATP at different concentrations: 0mmol/L, 1.6mmol/L, 2mmol/L, 2.5 mmol/L; (3) MgSO of varying total concentrations ₄ : 8mmol/L, 10mmol/L, 12 mmol/L. The three variables were used to set up crossover experiments to obtain 64 positive experimental groups (see tables 7 and 8), each group being 25. mu.l.

The positive experimental groups are all required to be provided with negative controls, and the negative controls consist of equal ddH ₂ And (4) replacing the template by O, and obtaining 64 negative control groups (NTC) if the other conditions are consistent with those of the positive experimental groups.

3. Loop-mediated isothermal amplification:

(1) configuration of LAMP reaction System (25. mu.l):

the reaction system was formulated as in table 4, with the difference that: without UvrD enzyme, adding proper amount of MgSO ₄ ATP and the template are added until the concentration of the ATP and the template in the corresponding system in the table 7 or the table 8 is met, the primers corresponding to GP130 respectively and correspondingly replace SEQ NO. 1-5 into SEQ NO. 6-10, and ddH is used for reducing the volume ₂ And (4) complementing O.

All the positive controls described above need to be provided with negative controls, and only 5 μ l of water is used to replace the corresponding template.

(2) Loop-mediated isothermal amplification: and (3) placing the reaction tube with the reaction system in a qPCR instrument, and reacting for 1h at the constant temperature of 63 ℃.

(3) Calculation of Ct value: after the reaction was completed, the Ct value of the reaction was calculated, and the results are shown in tables 7 and 8.

TABLE 7 ATP/MgSO 130 as template ₄ Concentration Cross-screening experiment

TABLE 8 ATP/MgSO4 concentration Cross-screening assay using lambda DNA as template

Finally, it should be noted that the above-mentioned embodiments only show a part of the results to illustrate the technical solutions of the present invention, but not to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Nanjing Novozan Biotechnology GmbH

Nanjing Novozam animal health care Co Ltd

New application of <120> ATP in LAMP, LAMP amplification system and kit

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Claims

Use of ATP in LAMP to inhibit non-specific amplification.
2. Use according to claim 1, wherein the concentration of ATP is 0.1-3 mmol/L, preferably 1.5-2.5 mmol/L.
3. Use according to claim 1, wherein the amplification template is a DNA template, preferably lambda DNA or GP 130.
4. An LAMP amplification system, which is characterized in that: the system comprises: ATP, nucleic acid template, primer, nucleic acid polymerase, buffer solution, dNTPs and magnesium sulfate.
5. The amplification system of claim 4, wherein: the system further includes a helicase selected from the group consisting of UvrD enzyme.
6. The amplification system according to claim 4, wherein: the nucleic acid template is a DNA template.
7. The amplification system of claim 6, wherein: 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.
8. The amplification system of claim 6, wherein: 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.
9. The amplification system of claim 6, wherein: the nucleic acid polymerase is DNA polymerase or its functional active fragment, preferably Bst DNA polymerase or its functional active fragment.
10. The amplification system of claim 6, wherein: 0.1-3 mmol/L of ATP, and the primer comprises: upstream inner primers of 0.5-2.5 mu mol/L, downstream inner primers of 0.5-2.5 mu mol/L, upstream outer primers of 0.05-0.4 mu mol/L, downstream inner primers of 0.05-0.4 mu mol/L and loop primers of 0.4-1.2 mu mol/L; the nucleic acid polymerase is 4-10U/mu L, the magnesium sulfate is 3.9-11 mmol/L, and the DNA template is 4-4 x10 ⁶ copies/. mu.l; the pH of the buffer solution is 7.5-9.4, and the buffer solution comprises: Tris-HCl 10-40 mmol/L, KCl 4-20 mmol/L, (NH) ₄ ) ₂ SO ₄ 4～20mmol/L，MgSO ₄ 0.1-5 mmol/L, Triton X-1000.01 w/w% -1 w/w%; the dNTPs comprise: 0.1-3 mmol/L of dATP, 0.1-3 mmol/L of dGTP, 0.1-3 mmol/L of dCTP and 0.1-3 mmol/L of dTTP.
11. The amplification system of claim 6, wherein: the ATP is 1.5-2.5 mmol/L, and the primer comprises: 1.0-2.0 mu mol/L of upstream inner primers, 1.0-2.0 mu mol/L of downstream inner primers, 0.1-0.3 mu mol/L of upstream outer primers, 0.1-0.3 mu mol/L of downstream inner primers and 0.6-1.0 mu mol/L of loop primers; 5-9U/mu L of nucleic acid polymerase, 5-10 mmol/L of magnesium sulfate and 4x10 of DNA template ¹ ～4x10 ⁵ copies/. 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-1000.05 w/w% -0.5 w/w%; the dNTPs comprise: 0.5-2 mmol/L of dATP, 0.5-2 mmol/L of dGTP, 0.5-2 mmol/L of dCTP and 0.5-2 mmol/L of dTTP.
12. The amplification system of claim 6, wherein: the ATP is 1.6mmol/L, and the primers comprise: upstream inner primer 1.6 mu mol/L, downstream inner primer 1.6 mu mol/L, upstream outer primer 0.2 mu mol/L, downstream inner primer 0.2 mu mol/L, and loop primer 0.8 mu mol/L; the nucleic acid polymerase6-8U/mu L, 6mmol/L magnesium sulfate and 4x10 DNA template ² ～4x10 ⁴ copies/. mu.l; the buffer has a pH of 8.8 and comprises: Tris-HCl 20mmol/L, KCl 10mmol/L, (NH) ₄ ) ₂ SO ₄ 10 mmol/L，MgSO ₄ 2mmol/L, Triton X-1000.1 w/w%; the dNTPs comprise: 1-1.4 mmol/L of dATP, 1-1.4 mmol/L of dGTP, 1-1.4 mmol/L of dCTP and 1-1.4 mmol/L of dTTP.
13. A LAMP amplification kit comprising the amplification system according to any one of claims 4 to 12.
14. A method for performing LAMP amplification detection using the kit of claim 13, comprising the steps of:

1) mixing ATP, primers, nucleic acid polymerase, 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 (5) carrying out isothermal amplification reaction on the mixed solution for detection.
15. The method of claim 14, wherein: the temperature range of the constant temperature amplification reaction is 30-75 ℃, preferably 50-70 ℃, and more preferably 60-65 ℃.