CN115786469A - Reagent for LAMP amplification reaction and preparation method thereof - Google Patents
Reagent for LAMP amplification reaction and preparation method thereof Download PDFInfo
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- CN115786469A CN115786469A CN202211461462.4A CN202211461462A CN115786469A CN 115786469 A CN115786469 A CN 115786469A CN 202211461462 A CN202211461462 A CN 202211461462A CN 115786469 A CN115786469 A CN 115786469A
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Abstract
The invention provides a self-research TRE reagent for LAMP amplification reaction and a preparation method thereof. The method mainly comprises the following steps: 1) The components are added in the following sequence: KCl, (NH 4) 2 SO 4 、MgSO 4 Adding deionized water into the phenol red solution, triton X-100 or Tween 20, and adjusting the pH value to obtain a mixed solution; 2) The components are added in the following order: deionized water, the mixed solution obtained in the step 1), DNTP and 2.0DNA polymerase. Compared with the existing reagents in the market, the self-developed TRE reagent prepared by the method has the following advantages: the process for preparing the reaction system is optimized, the system error caused by batch effect in the production process is reduced, and the reaction accuracy is improved; the concentration ratio of reaction components of the traditional LAMP system is optimized, and the influence of a color indicator on LAMP reaction is reduced by adopting a low-concentration phenol red solution as the color indicator, so that the amplification efficiency is improved; the pH value of the reaction system is reduced, the sensitivity of the reaction system is improved, and the enzyme is more stable compared with the existing NEB enzymeAnd under the condition of unchanged efficiency, the cost is reduced by more than 10 times.
Description
Technical Field
The invention relates to the field of biological experiment reagents, in particular to a reagent for LAMP amplification reaction and a preparation method thereof.
Background
The loop-mediated isothermal amplification method is a novel nucleic acid amplification method and has the characteristics of simple operation, strong specificity, easy detection of products and the like. Upon DNA synthesis, pyrophosphate ions precipitated from deoxyribonucleic acid triphosphate substrates (dNTPs) reacted with magnesium ions in the reaction solution to generate a large amount of magnesium pyrophosphate precipitate, which appeared white. Therefore, the turbidity can be used as an index of the reaction, and whether amplification is carried out or not can be identified only by observing the white turbid precipitate with naked eyes without complicated electrophoresis and ultraviolet observation. Because the loop-mediated isothermal amplification reaction does not need a PCR instrument and expensive reagents, the method has wide application prospect.
The pH indicator color development is a common detection scheme of the LAMP reagent, however, in the reagent preparation process, as the LAMP reagent based on the pH indicator color development has poor buffering capacity and different components need to be added separately, the operation is complicated, the batch effect is extremely large, and the color development effects of different batches of reagents have obvious difference.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a bufferless LAMP amplification reagent TRE reddish yellow discoloration 2 × LAMP MIX reagent and a preparation method thereof, which are used for solving the disadvantages of low sensitivity and poor stability in the prior art. The low sensitivity means that the reaction time of the existing reagent is longer at 40-60 min; the problem of large experimental error is caused by incomplete consistency of pH indication end points among different batches due to the fact that the formula and the preparation method in the prior art are not scientific enough. The invention improves the two aspects simultaneously, firstly, the reaction component concentration of the traditional LAMP system is optimized, the chromogenic indicator with lower concentration and low pH are adopted, the influence of the chromogenic indicator on the LAMP reaction is reduced, the amplification efficiency is improved, the reagent sensitivity is increased, and the reaction time is reduced to be within 15 minutes from 40-60 min; secondly, the reagent preparation method is optimized, domestic raw materials are scientifically proportioned, and the cost is reduced by more than 10 times on the basis of improving the efficiency. And due to the optimization of the production flow, the stability of products in different batches is enhanced.
The invention provides a preparation method of a reagent for LAMP amplification reaction, which comprises the following steps:
1) The components are added in the following sequence: KCl, (NH 4) 2 SO 4 、MgSO 4 Adding deionized water into the phenol red solution, triton X-100 or Tween 20, and adjusting the pH value to obtain a mixed solution;
2) The components are added in the following order: deionized water, the mixed solution obtained in the step 1), DNTP and BST DNA polymerase.
Further, the final concentration of KCl in the step 1) is 300-800mM; (NH 4) 2 SO 4 Is 100-200mM, mgSO 4 The final concentration of (A) is 10-20mM, the final concentration of phenol red in the phenol red solution is 100-500mM, and the final concentration of Triton X-100 is 0.1-0.5%.
Further, the final concentration of KCl in the step 1) is 500mM; (NH 4) 2 SO 4 Is 100mM, mgSO 4 The final concentration of (A) was 12mM, the final concentration of phenol red in the phenol red solution was 300mM, and the final concentration of Triton X-100 was 0.50% (mass/volume).
Further, the pH is adjusted to 8 to 8.5, preferably 8.4 in the step 1).
Further, the step 1) further comprises shaking and uniformly mixing the prepared mixed solution. The technicians in the field can adopt the oscillation mixing mode in the prior art to mix evenly, and can adopt a shaking table or an ultrasonic mixing mode; the preferred mode is ultrasonic blending.
Further, the step 1) further comprises sterilizing the mixed solution after shaking; the preferred mode is filter sterilization.
Further, the volume ratio of each component in the step 2) is deionized water: obtaining a mixed solution in the step 1): DNTP: BST2.0 DNA polymerase = 2.5.
According to another aspect of the invention, a TRE reddish yellow discolor 2 LAMP MIX reagent for LAMP amplification reaction is provided, and the reagent is prepared by adopting the method.
In another aspect of the present invention, there is provided a use of the above-described reagent for LAMP amplification reaction.
As described above, the reagent for LAMP amplification reaction of the present invention has the following advantageous effects:
the prepared reagent optimizes the concentration of reaction components of the traditional LAMP system, adopts a lower-concentration chromogenic indicator and a low pH value, reduces the influence of the chromogenic indicator on LAMP reaction, improves the amplification efficiency, increases the sensitivity of the reagent and reduces the reaction time from 40-60min to within 15 min; secondly, the reagent preparation method is optimized, domestic raw materials are scientifically proportioned, and the cost is reduced by more than 10 times on the basis of improving the efficiency. And due to the optimization of the production flow, the stability of products in different batches is enhanced.
Reference numerals
FIG. 1 shows the color difference of our developed reaction solution and NEB imported enzyme for different batches at the same time.
FIG. 2 shows the color change of the reaction solution developed by us after heating with the NEB inlet enzyme.
FIG. 3 shows the electrophoresis photograph of the product obtained by LAMP reaction of the reaction solution developed by me and NEB import enzyme.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art. Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is only a convenient tool for identifying each method step, and is not intended to limit the order of the method steps or the scope of the invention, and changes or modifications in the relative relationship thereof may be regarded as the scope of the invention without substantial change in the technical content.
Experimental materials and instruments：
An experimental instrument: deionized water pure water system, horizontal shaking table, pH detector
Consumable material: 0.22 μ M aqueous phase needle filter, 10ml syringe
EXAMPLE 1 preparation of stain use solution
Dye storage liquid: weighing 17.7mg of phenol red powder into a 2ml EP tube, adding 400 mu L of DMSO, shaking and mixing uniformly until the phenol red powder is completely dissolved, and storing at-20 ℃.
Coloring agent use liquid: 250 μ L of the phenol red DMSO solution is put into a 50mL test tube, deionized water is added to the test tube to reach 50mL, and a stain solution with a final concentration of 500 μ M is obtained.
Example 2 LAMP reaction inductive buffer preparation
Prepare 10 × lamp reaction sensitive buffer as in table 1: adding the components in the sequence in the table for preparation, and then placing the mixed solution in an ultrasonic oscillator for oscillation for 5-10min (power is 40%, temperature is 25 ℃) to ensure that the precipitate in the mixed solution is completely dissolved to obtain a transparent orange yellow solution. The prepared solution is filtered and sterilized by a 0.22 mu M aqueous phase filtering membrane, then subpackaged and stored at the temperature of minus 20 ℃ for a long time, and the LAMP reaction effect is not influenced when the solution is stored at the room temperature for about two weeks under the condition of no pollution. (Note: 1. After preparation, vigorous shaking is prohibited, otherwise large amount of bubbles will be generated; 2. Tween 20 can be used instead of Triton X-100.) Table 1 LAMP reaction inductive buffer formula 10
Example 3lamp MIX formulation
Preparing 2 LAMP MIX according to table 2, adding from top to bottom according to the sequence in the table, shaking, mixing uniformly and centrifuging instantly after the preparation is finished.
Table 2 lamp MIX formulations
Example 4 reagents for LAMP amplification reaction
Taking a small amount of self-developed TRE reddish yellow discoloring 2 LAMP MIX reagent, preparing an LAMP reaction system according to the table 3, shaking, uniformly mixing and instantaneously centrifuging, using NEB LAMP reaction 2 MIX as a control, and carrying out comparative test on the following indexes:
if the self-developed TRE reddish yellow discoloration 2 × LAMP MIX reagent is compared with an LAMP reaction system prepared from the NEB reagent, the color change before and after the reaction is similar, and the DNA electrophoresis products are similar, the reagent is considered to be successfully prepared.
TABLE 3LAMP reaction System
1. And (3) comparing the color of the LAMP reaction system with that of a reaction system prepared by NEB reagent before heating: the purchased NEB reagent has large color difference among different batches as shown in figure 1, the colors of different batches of the conventional NEB reagent have three color states of dark purple, light purple and red saving, the experimental results are judged by the color change of the reagent, so that the experimental results obtained by the NEB reagents of different batches are unstable, the experimental results of different batches cannot be mutually compared, the accuracy of the experimental results is greatly influenced, the application of LAMP technology in the field of biomedicine is limited, the TRE reagent developed by people has no color difference among different batches, the experimental results can be mutually compared, and the problem is solved.
2. Heating the two systems in water bath at 65 deg.C for 15min, and comparing colors;
as shown in fig. 2, after the NEB reagent is heated in a water bath at 65 ℃ for 15min, the color of the reagent does not change, because the color change reaction does not reach the critical point, and the self-developed TRE reddish yellow discolor 2 × lamp MIX reagent has obviously changed, which indicates that the self-developed TRE reddish yellow discolor 2 × lamp MIX reagent has higher sensitivity.
3. The LAMP reaction solution after the reaction was subjected to electrophoresis using 1.5% agarose gel in 5. Mu.L, and the DNA bands of the reaction products were compared, and the results are shown in FIG. 3. The electrophoresis bands generated by the two reagents are the same, which indicates that LAMP amplification reaction occurs, but only TRE red-yellow discoloration 2. LAMP MIX changes the color at 15 min.
The results show that the developed reaction solution can distinguish negative samples from positive samples, and the self-developed TRE reddish yellow discoloration 2 × LAMP MIX reagent has higher sensitivity.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention is not limited to those specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (9)
1. A method for preparing a reagent for a LAMP amplification reaction, the method comprising at least the steps of:
1) The components are added in the following sequence: KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Adding deionized water into the phenol red solution, triton X-100 or Tween 20, and adjusting the pH value to obtain a mixed solution;
2) The components are added in the following order: deionized water, the mixed solution obtained in the step 1), DNTP and Bst2.0 DNA polymerase.
2. The method for preparing a reagent for LAMP amplification reaction according to claim 1, characterized in that: the final concentration of KCl in the step 1) is 300-800mM; (NH 4) 2 SO 4 Has a final concentration of 100-200mM, mgSO 4 The final concentration of (A) is 10-20mM, the final concentration of phenol red in the phenol red solution is 100-500mM, and the final concentration of Triton X-100 is 0.1-0.5%.
3. The method of preparing a reagent for LAMP amplification reaction according to claim 1, characterized in that: adjusting the pH value to 8-8.5 in the step 1).
4. The method for preparing a reagent for LAMP amplification reaction according to claim 1, characterized in that: adjusting the pH value to 8.4 in the step 1); the final concentration of KCl is 500mM; (NH) 4 ) 2 SO 4 Is 100mM, mgSO 4 Was 12mM, and the final concentration of phenol red in the phenol red solution was 300mM.
5. The method for preparing a reagent for LAMP amplification reaction according to claim 1, characterized in that: the step 1) also comprises shaking and uniformly mixing the prepared mixed solution.
6. The method for preparing a reagent for LAMP amplification reaction according to claim 5, characterized in that: the step 1) also comprises the step of sterilizing the mixed solution after shaking.
7. The method for preparing a reagent for LAMP amplification reaction according to claim 1, characterized in that: the volume ratio of each component in the step 2) is deionized water: obtaining a mixed solution in the step 1): DNTP: BST DNA polymerase = 2.5.
8. A reagent prepared by the method of any one of claims 1 to 7.
9. Use of the reagent of claim 8 for a LAMP amplification reaction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116042785A (en) * | 2023-03-22 | 2023-05-02 | 翌圣生物科技(上海)股份有限公司 | Buffer for RT-LAMP amplification reagent and RT-LAMP amplification reagent |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116042785A (en) * | 2023-03-22 | 2023-05-02 | 翌圣生物科技(上海)股份有限公司 | Buffer for RT-LAMP amplification reagent and RT-LAMP amplification reagent |
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