LAMP (loop-mediated isothermal amplification) visual detection color reagent and application thereof

Abstract

The invention provides an LAMP (loop-mediated isothermal amplification) visual detection chromogenic reagent and application thereof. The chromogenic reagent is a double chromogenic reagent and is used for indicating whether amplification occurs before and after an LAMP reaction, and whether the reaction occurs is judged by observing negative and positive color changes of the LAMP reaction, so that visual detection of LAMP is realized. The color-developing reagent has a remarkable and distinct color-changing effect, so that the LAMP detection result is clear at a glance, and the color-developing reagent has the characteristics of sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid molecule detection and analysis, and more specifically, relates to a type of visualized detection color developing reagent for loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology and its application. Background Art

[0002] Japanese scholar Notomi published a new isothermal nucleic acid amplification technology suitable for genetic diagnosis in the journal Nucleic Acids Res (2000), namely loop-mediated isothermal amplification (LAMP). The reaction system generally includes 4 primers (2 outer primers and 2 inner primers), Bst DNA polymerase buffer, Bst DNA polymerase with chain displacement properties, dNTP, template DNA, Mg2SO4, etc. LAMP has high amplification efficiency and specificity. It can amplify the template by 10 within 30 to 60 minutes at a temperature of 60 to 65°C. 9 ~10 10 times. In 2002, Nagamine significantly accelerated the speed of isothermal amplification (<30min) and greatly improved the detection efficiency by adding loop primers. Therefore, the current LAMP amplification experiment mainly uses a combination of 6 primers, including two outer primers (F3 and B3), two inner primers (FIP (F1c + F2) and BIP (B1c + B2)), and two loop primers (LoopF ​​and LoopB). If the sensitivity requirement is not high, a four-primer combination without LoopF / B can still be used.

[0003] The LAMP method has been used to detect a variety of pathogens in clinical, food and environmental samples, such as bacteria, viruses, parasites and protozoa. There are many ways to detect its amplified products, and it is constantly developing in a more accurate, simple and efficient direction.

[0004] LAMP amplification products can be detected by agarose gel electrophoresis. After LAMP amplification, a large number of DNA mixtures with different lengths, different numbers of stem-loop structures and inverted repeat sequences can be produced, and agarose gel electrophoresis presents waterfall-like ladder bands. The disadvantage of electrophoresis detection is that the amplification tube needs to be opened, and high-concentration DNA products are prone to produce aerosols, causing laboratory environmental pollution.

[0005] Mori et al. proposed a method for identifying LAMP amplification by turbidity analysis in a closed system in 2001. Due to the high efficiency of LAMP reaction, the amount of DNA can be amplified by 10 within 15 to 60 minutes. 9 ~1010 When nucleic acids are synthesized in large quantities, the pyrophosphate ions precipitated from dNTPs combine with the magnesium ions in the reaction buffer to produce a byproduct, magnesium pyrophosphate, forming a milky white precipitate. Moreover, there is a certain correlation between the yield of magnesium pyrophosphate and the concentration of the amplified product. Therefore, after the amplification is completed, the milky white precipitate may be observed by the naked eye. Generally, a turbidity meter is used to monitor the change of turbidity in real time.

[0006] Compared with the PCR method, LAMP does not require a thermal cycler (PCR instrument). For example, a large amount of by-products - white magnesium pyrophosphate precipitates - can be produced in the LAMP reaction, and the amplification products can be observed by the naked eye or with a turbidity meter to determine the results; therefore, LAMP is suitable for rapid detection on-site or in laboratories with poor conditions.

[0007] According to the literature, there are also reports of attempts to detect LAMP amplification products using instrument-independent methods. Depending on whether the dye inhibits the reaction, the dye can be added after or before the reaction. There are many types of dyes, and three of them are more commonly used: SYBR Green, calcein, and hydroxynaphthol blue (HNB). SYBR Green inhibits LAMP reactions and needs to be added after the reaction. Opening the lid can easily cause environmental pollution; calcein can be added before the reaction, but it will reduce the sensitivity of LAMP; HNB can also be added before the reaction. HNB and Mg before the reaction should be added. 2+ The two molecules combine to form a violet color. When the DNA double chain is synthesized, magnesium pyrophosphate is generated. The HNB that loses the magnesium ion becomes sky blue. Currently, some companies have applied this principle to develop commercial reagents, but HNB is unstable and difficult to preserve. Another problem is that the color reaction of HNB is not obvious to the naked eye, and the color change is not easy to detect.

[0008] Therefore, LAMP detection technology still needs further technical optimization in order to further improve its convenience and accuracy of detection. Summary of the invention

[0009] The purpose of the present invention is to provide a type of visualized detection color developing reagent for aptamer loop mediated isothermal amplification technology (LAMP) and its application.

[0010] In a first aspect of the present invention, a LAMP visualization detection method is provided, comprising using a visualization colorimetric reagent group in a LAMP reaction system; the dual display reagent comprises a colorimetric reagent 1 and a colorimetric reagent 2; the colorimetric reagent 1 changes from red to yellow within a range of 8.4±0.4 (preferably 8.4±0.3 or 8.4±0.2 or 8.4±0.1) to 6.8±0.2; the colorimetric reagent 2 comprises a blue, green or purple colorimetric reagent, which increases the color difference of the colorimetric reagent 1 and enhances the visualization analysis of the LAMP reaction product.

[0011] In one or more embodiments, the color developing reagent 1 includes: phenol red or cresol red, or a combination thereof; the color developing reagent 2 includes: methylene blue, bromocresol green, crystal violet, fast green or hydroxynaphthol blue, or a combination thereof.

[0012] In one or more embodiments, the visualization color developing reagent group includes: phenol red and methylene blue, phenol red and bromocresol green, phenol red and crystal violet, phenol red and fast green, phenol red and hydroxynaphthol blue; cresol red and methylene blue, cresol red and bromocresol green, cresol red and crystal violet, cresol red and fast green, cresol red and hydroxynaphthol blue.

[0013] In one or more embodiments, in the visualization color reagent set:

[0014] Phenol red:methylene blue is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1;

[0015] Phenol red: bromocresol green is 1:1 to 4:1, preferably 1.5:1 to 3.5:1, more preferably 1.5:1 to 3:1 (e.g. 2:1, 2.5:1);

[0016] Phenol red: crystal violet is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1;

[0017] Phenol red:fast green is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1;

[0018] Phenol red:hydroxynaphthol blue is 0.5:1-3:1, preferably 1:1-2.5:1, more preferably 1:1-2:1 (such as 1.5:1);

[0019] Cresol red:methylene blue is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1;

[0020] Cresol red: bromocresol green is 0.5:1 to 4:1, preferably 1:1 to 3.5:1, more preferably 1.5:1 to 3:1 (such as 2:1, 2.5:1);

[0021] Cresol red: crystal violet is 1.5:1-4:1, more preferably 1.5:1-3:1, more preferably 2:1-3:1 (such as 2.5:1);

[0022] Cresol red:fast green is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1;

[0023] The ratio of cresol red to hydroxynaphthol blue is 0.5:1 to 3:1, more preferably 1:1 to 2.5:1, more preferably 1:1 to 2:1 (such as 1.5:1).

[0024] In one or more embodiments, when performing the LAMP reaction, a sample to be tested containing a nucleic acid template is added to the LAMP reaction system; if the nucleic acid template contains a target nucleic acid that can be recognized and amplified by the primer, the visual color development reagent set undergoes a color change (a significant / bright color change).

[0025] In one or more embodiments, the final concentration of the color developing reagent 1 or the color developing reagent 2 in the LAMP reaction system is 50-200 μM (such as 60, 80, 120, 140, 150, 160 μM); preferably 100-180 μM.

[0026] In one or more embodiments, the LAMP reaction system includes (mainly includes): a visualization color development reagent set, dNTPs, DNA polymerase, MgSO4, and a reaction buffer; and optionally also includes a LAMP primer set.

[0027] In one or more embodiments, the visualization colorimetric reagent set is phenol red and methylene blue, and the color changes include: purple→green (i.e., purple when there is no amplification and green when there is amplification), blue→green (i.e., blue when there is no amplification and green when there is amplification), or purple→pink (i.e., purple when there is no amplification and pink when there is amplification).

[0028] In one or more embodiments, the visualization colorimetric reagent set is phenol red and bromocresol green, and the color changes include: purple→green (i.e., purple when there is no amplification and green when there is amplification), blue→green (i.e., blue when there is no amplification and green when there is amplification), purple→green (i.e., purple when there is no amplification and green when there is amplification), or pink→orange (i.e., pink when there is no amplification and orange when there is amplification).

[0029] In one or more embodiments, the visualization color reagent set is phenol red and crystal violet, and the color change includes: purple-red→brown (ie: purple-red without amplification and brown with amplification).

[0030] In one or more embodiments, the visualization colorimetric reagent set is phenol red and fast green, and the color changes include: (pink) red→(light) green (i.e., (pink) red when there is no amplification, (light) green when there is amplification) or orange→yellow (i.e., orange when there is no amplification, yellow when there is amplification).

[0031] In one or more embodiments, the visualization color developing reagent group is phenol red and hydroxynaphthol blue, and the color changes include: blue→yellow (i.e., blue when there is no amplification and yellow when there is amplification), purple→green (i.e., purple when there is no amplification and green when there is amplification), purple-red→green (i.e., purple-red when there is no amplification and green when there is amplification), rose-red→brown (i.e., rose-red when there is no amplification and brown when there is amplification), (light) pink→brown (i.e., (light) pink when there is no amplification and brown when there is amplification).

[0032] In one or more embodiments, the visualization colorimetric reagent set is cresol red and methylene blue, and the color changes include: blue→green (i.e., blue when there is no amplification and green when there is amplification), purple→green (i.e., purple when there is no amplification and green when there is amplification), or reddish brown→green (i.e., reddish brown when there is no amplification and green when there is amplification).

[0033] In one or more embodiments, the visualization color developing reagent set is cresol red and bromocresol green, and the color changes include: blue→green (i.e., blue when there is no amplification and green when there is amplification), purple→green (i.e., purple when there is no amplification and green when there is amplification), purple-red→yellow-green (i.e., purple-red when there is no amplification and yellow-green when there is amplification), or pink→orange (i.e., pink when there is no amplification and orange when there is amplification).

[0034] In one or more embodiments, the visualization colorimetric reagent set is cresol red and crystal violet, and the color changes include: purple-red→reddish brown (i.e., purple-red without amplification and reddish brown with amplification), or purple-red→brown (i.e., purple-red without amplification and brown with amplification).

[0035] In one or more embodiments, the visualization color developing reagent group is cresol red and fast green, and the color changes include: (light) pink→green (i.e., (light) pink when there is no amplification and green when there is amplification), pink→green (i.e., pink when there is no amplification and green when there is amplification), purple→green (i.e., purple when there is no amplification and green when there is amplification), pink→yellow (i.e., pink when there is no amplification and yellow when there is amplification), or rose→orange (i.e., rose when there is no amplification and orange when there is amplification).

[0036] In one or more embodiments, the visualization colorimetric reagent group is cresol red and hydroxynaphthol blue, and the color changes include: blue→green (i.e., blue when there is no amplification and green when there is amplification), purple→green (i.e., purple when there is no amplification and green when there is amplification), brown→yellow (i.e., brown when there is no amplification and yellow when there is amplification), orange→yellow (i.e., orange when there is no amplification and yellow when there is amplification).

[0037] In one or more embodiments, the color change is analyzed by setting a test group and a control group.

[0038] In another aspect of the present invention, a LAMP visualization detection reagent is provided, which is a visualization colorimetric reagent group, including a colorimetric reagent 1 and a colorimetric reagent 2; the colorimetric reagent 1 undergoes a change from red to yellow at 8.4±0.4 (preferably 8.4±0.3 or 8.4±0.2 or 8.4±0.1); the colorimetric reagent 2 includes a blue, green or purple colorimetric reagent, which increases the color difference of the colorimetric reagent 1 and enhances the visualization analysis of the LAMP reaction product.

[0039] In one or more embodiments, the color developing reagent 1 includes: phenol red or cresol red, or a combination thereof; the color developing reagent 2 includes: methylene blue, bromocresol green, crystal violet, fast green or hydroxynaphthol blue, or a combination thereof.

[0040] In one or more embodiments, the visualization color developing reagent group includes: phenol red and methylene blue, phenol red and bromocresol green, phenol red and crystal violet, phenol red and fast green, phenol red and hydroxynaphthol blue; cresol red and methylene blue, cresol red and bromocresol green, cresol red and crystal violet, cresol red and fast green, cresol red and hydroxynaphthol blue.

[0041] In another aspect of the present invention, a reaction system for LAMP visualization detection is provided, which comprises the LAMP visualization detection reagent.

[0042] In one or more embodiments, the reaction system also includes (mainly includes): a visualization color development reagent set, dNTPs, DNA polymerase, MgSO4, and a reaction buffer; and optionally also includes a LAMP primer set.

[0043] In another aspect of the present invention, the application of the LAMP visualization detection reagent and the reaction system is provided for performing LAMP visualization detection, or for preparing a detection system or kit for performing LAMP visualization detection.

[0044] In another aspect of the present invention, a LAMP visualization detection kit is provided, comprising: the LAMP visualization detection reagent; or the reaction system for LAMP visualization detection.

[0045] In one or more embodiments, the detected reagent or reaction system includes a liquid system, such as a solution system, or a solid system (including a freeze-dried system).

[0046] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 In Example 1, HPV16 or HPV18 is used as a template and a dual color reagent is used to participate in the LAMP reaction, and the colors of the negative and positive reaction tubes.

[0048] Figure 2 Example 1: Using HPV16 or HPV18 as a template and utilizing a single color developing reagent to participate in the LAMP reaction, the colors of the negative and positive reaction tubes.

[0049] Figure 3 , color development of phenol red and methylene blue in different proportions.

[0050] Figure 4 , color development of phenol red and bromocresol green in different proportions.

[0051] Figure 5 , color development of phenol red and crystal violet in different proportions.

[0052] Figure 6 , color development of phenol red and fast green in different proportions.

[0053] Figure 7 , the dual color reagent can be stored for a long time (12 months at room temperature) for stability analysis.

[0054] Figure 8 , liquid and dry mixed colorimetric reagents for application analysis.

[0055] Fig. 9 , cresol red and methylene blue combination in different proportions.

[0056] Fig.10 , cresol red and bromocresol green in different proportions to show color development.

[0057] Fig.11 , cresol red and crystal violet in different proportions.

[0058] Fig.12 , cresol red and fast green combination in different proportions.

[0059] Fig.13 , hydroxynaphthol blue and phenol red mixed color development.

[0060] Fig.14 , hydroxynaphthol blue and cresol red mixed color development. DETAILED DESCRIPTION

[0061] After in-depth research and experiments, the inventors have revealed a type of visual detection color reagent suitable for loop-mediated isothermal amplification technology (LAMP), which is a dual color reagent that indicates whether amplification occurs before and after the LAMP reaction. By observing the color changes of negative and positive LAMP reactions, it is judged whether the reaction occurs, thereby realizing visual detection of LAMP. The color change effect of the color reagent is significant and distinct, so that the result of the LAMP detection can be determined at a glance, and it has the characteristics of sensitivity and accuracy.

[0062] As used in the present invention, the "color developing reagent", "staining reagent", "staining agent" and "dye" can be used interchangeably.

[0063] As used in the present invention, the "visualization color developing reagent (set)", "dual color developing reagent (set)", "reagent combination" and the like can be used interchangeably.

[0064] As used in the present invention, the "sample to be tested" is not particularly limited and can be (but not limited to) a variety of samples suitable for detection using the LAMP technology. The "sample to be tested" may contain nucleic acid (such as DNA) to analyze whether the target nucleic acid exists in the nucleic acid using the LAMP technology.

[0065] As used in the present invention, "target nucleic acid" is a nucleic acid that is the object of analysis and characterization of the technical solution of the present invention. For example, in a sample containing genomic DNA of a virus / bacteria that is the object of measurement, genomic DNA of a virus / bacteria that is not the object of measurement, and other DNA, the target nucleic acid is a nucleic acid derived from the virus / bacteria that is the object of measurement. The objects can be broad, such as microorganisms (viruses or bacteria, etc.), animals, plants, environmental samples (such as samples from public places, utensils, soil, river water, sea water, hot spring water, etc.), or some objects containing them or their processed products.

[0066] The method for obtaining nucleic acid from the sample to be tested is a technique well known to those skilled in the art, for example, the traditional phenol / chloroform / isoamyl alcohol method can be adopted, or some commercially available nucleic acid extraction kits can be used, such kits are well known to those skilled in the art.

[0067] In the present invention, the visualization color reagent is a reagent set, including a color reagent 1 and a color reagent 2. The color reagent 1 changes from red to yellow within a range of 8.4±0.2 to 6.8±0.2; the color reagent 2 includes a blue, green or purple color reagent, which increases the color difference of the color reagent 1 and enhances the visualization analysis of the LAMP reaction product.

[0068] In the LAMP reaction process with amplification, a large number of protons are generated, which causes the pH value in the system to decrease before and after the reaction. A visual color developing reagent is added to the reaction system, and the color change before and after the reaction can be used to judge whether the LAMP reaction is amplified. The present invention adopts a method of mixing two color developing reagents, one color developing reagent undergoes a suitable color change within the pH change range of the LAMP reaction, and the other color developing reagent enhances the color difference before and after the reaction. The two synergize and greatly optimize the interpretation of the LAMP detection results. The present invention provides an optimized visual color developing reagent, which makes the color change more prominent.

[0069] In a preferred embodiment of the present invention, the visual color developing reagent is selected from the following reagents: phenol red, cresol red, methylene blue, bromocresol green, crystal violet, fast green or hydroxynaphthol blue. Methylene blue, phenol red, fast green, crystal violet, bromocresol green are generally used as staining agents or indicators (Table 1). Among the color developing reagents used in the previous LAMP technology, SYBR Green, calcein or hydroxynaphthol blue (HNB) are mostly used. The present invention is very different from the previous technology.

[0070] Table 1. General uses of colorimetric reagents

[0071]

[0072]

[0073] In a preferred embodiment of the present invention, after the two color developing reagents are mixed, exemplary amplification colors are shown in Table 2.

[0074] Table 2

[0075] usage Color development reagent 1 Chromogenic reagent 2 Dosage No amplified color Amplified colors 1 Methylene blue Phenol red 50~180μM Purple green 2 Phenol red Bromocresol green 50~180μM Purple green 3 Crystal violet Phenol red 50~180μM Purple brown 4 Phenol red Fast Green 50~180μM red Light green

[0076] The color developing reagent of the present invention can be used in combination with a commercial liquid reagent, and can also be used in combination with a commercial freeze-dried reagent.

[0077] The color developing reagent of the present invention has relatively stable properties and can be used as a liquid mix or after being dried without affecting the reaction performance.

[0078] The present invention also provides a reaction system for LAMP visual detection, which can be pre-prepared or prepared during the experiment. The reaction system includes the visual color development reagent set. In a preferred embodiment, the reaction system also includes: a visual color development reagent set, dNTPs, DNA polymerase, MgSO4, and a reaction buffer; optionally, a LAMP primer set is also included, and the LAMP primer set can be added during pre-preparation or during the experiment.

[0079] The LAMP reaction can be performed by methods known in the art or methods being improved. It should be understood that relatively stable pH changes will occur during the LAMP reaction, so the visualization color reagent set of the present invention is compatible with LAMP reactions for a variety of target nucleic acids and a variety of reaction systems for LAMP reactions, all of which can be included in the present invention.

[0080] In a preferred embodiment of the present invention, the general reaction system of the liquid reagent is exemplified as follows:

[0081]

[0082] After the reaction is completed, observe the colors of the negative sample and the positive sample. The color of the negative sample is purple, and the color of the positive sample is green.

[0083] The present invention also includes a LAMP detection kit containing the visualization color reagent, wherein the kit includes: the visualization color reagent set of the present invention, or the reaction system for LAMP visualization detection of the present invention. Other peripheral reagents commonly used in LAMP detection operations may also be included in the kit to facilitate the use of those skilled in the art. In addition, the kit may also include instructions for use to guide those skilled in the art to operate.

[0084] In a preferred embodiment, the kit, especially the instruction manual, also includes some instructions on the correlation between color changes and reaction results, so as to guide the staff to intuitively interpret the reaction results.

[0085] The main priorities of the present invention include:

[0086] (1) The visualized color developing reagent of the present invention has no inhibitory effect on the LAMP reaction within a certain concentration range and can be added before the reaction to avoid opening the lid after the reaction to avoid nucleic acid contamination.

[0087] (3) The visualized color developing reagent described in the present invention has high stability and is easy to store and use.

[0088] (4) The visualized color developing reagent of the present invention has obvious color difference when distinguishing whether amplification occurs in the LAMP reaction, which is conducive to direct observation with the naked eye.

[0089] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

[0090] Materials and methods

[0091] 1. LAMP reaction system

[0092] The color developing reagent of the present invention can be used in combination with a commercial liquid reagent, and can also be used in combination with a commercial freeze-dried reagent.

[0093] In the subsequent examples, unless otherwise stated, the general reaction system of liquid reagents is as follows:

[0094]

[0095] Among them, Bst DNA Polymerase, reaction buffer, dNTPs, and MgSO4 are all commercial reagents, and the reaction can be carried out by simply adding the dissolving solution, primers, and color developing reagent.

[0096] 2. Adaptable instruments

[0097] Water bath, metal bath, PCR instrument, etc.

[0098] 3. LAMP reaction conditions

[0099] 60~65℃;

[0100] After the reaction is completed, observe the colors of the negative and positive samples.

[0101] 4. Exemplary LAMP reaction template and exemplary LAMP reaction primer

[0102] In the following examples, unless otherwise stated, LAMP detection for HPV16 and HPV18 is taken as an example.

[0103] Template for LAMP reaction: HPV16 plasmid or HPV18 plasmid extracted using a commercial kit.

[0104] The sequences of the designed HPV16 and HPV18 primers are shown in Table 3.

[0105] Table 3

[0106]

[0107] Example 1: LAMP reaction analysis using different colorimetric reagents

[0108] 1. Color development effect of dual color reagent

[0109] A dual colorimetric reagent set was used, and the types and amounts of the colorimetric reagents are shown in Table 2.

[0110] HPV16 or HPV18 was used as a template, and the LAMP reaction system and reaction method in the aforementioned materials and methods were used for detection, and a dual color development reagent was added; sterile water (negative template) was used as a negative sample.

[0111] The color development results are shown in Table 4 and Figure 1 It can be seen that each group of samples presents a bright color, and there is a very significant color difference between the negative samples and the positive samples.

[0112] Table 4

[0113]

[0114] 2. Color development effect of single color reagent

[0115] A single color developing reagent set was used, and the color developing reagents included phenol red, methylene blue, bromocresol green, crystal violet, and fast green, and the amounts used are shown in Table 3.

[0116] HPV16 or HPV18 was used as a template, and the LAMP reaction system and reaction method in the aforementioned materials and methods were used for detection, and a single color developing reagent was added; a sample of sterile water was used as a negative sample.

[0117] The color development results are shown in Table 5 and Figure 2 It can be seen that when phenol red, methylene blue, bromocresol green, crystal violet and fast green are used separately in the LAMP system, phenol red and fast green can distinguish the negative samples from the positive samples after amplification by color, but the color distinction between negative and positive samples is low, and the other several have no function of distinguishing the negative and positive samples after amplification.

[0118] Table 5

[0119]

[0120]

[0121] Therefore, when a dual color reagent combination is used, the ability to distinguish between negative and positive samples after amplification can be greatly improved by distinguishing by color.

[0122] Example 2, analysis of the dosage of dual color reagent

[0123] The color development effect and color differentiation degree of the dual color development reagent combination at different dosages were analyzed. A dual color development reagent set was used, and the types and dosages of the color development reagents are shown in Table 6.

[0124] HPV16 plasmid was used as a template, and the LAMP reaction system and reaction method in the aforementioned materials and methods were used for detection, and a dual color development reagent was added; a sample of sterile water was used as a negative sample.

[0125] The color development results of different ratios of phenol red and methylene blue are shown in Table 6 and Figure 3 It can be seen that the colors of the positive sample and the negative sample can be identified as different under the ratio (1:1-4:1), among which the colors under 2:1-3:1 are the most distinct.

[0126] The color development results of different proportions of phenol red and bromocresol green are shown in Table 6 and Figure 4 It can be seen that the colors of the positive sample and the negative sample can be well identified as different under the ratio (1:1-4:1), among which the colors under 1.5:1-3:1 are the most distinct.

[0127] The color development results of different proportions of phenol red and crystal violet are shown in Table 6 and Figure 5 It can be seen that the color difference between the positive sample and the negative sample is most obvious at 2:1 to 3:1.

[0128] The color development results of different proportions of phenol red and fast green are shown in Table 6 and Figure 6 It can be seen that the colors of the positive sample and the negative sample can be well identified as different under the ratio (1:1-4:1), among which the colors under 2:1-3:1 are the most distinct.

[0129] Table 6. Color of the dual color reagent combination at different ratios

[0130]

[0131]

[0132] Therefore, the colors are somewhat different when the ratio of the two dyes is different. In the color development effect, when the ratio of the two dyes is within a certain range, the larger the color difference between the negative sample and the positive sample, the more conducive to naked eye identification.

[0133] Example 3, Stability Analysis of Dual Color Reagent

[0134] The color developing reagents are mixed and the stability of the dual color developing reagent is analyzed.

[0135] like Figure 7 As shown, the dual color reagent can be stored for a long time (12 months at room temperature) and its properties are relatively stable.

[0136] The dual color reagent can be prepared as a liquid mix or used after drying, which has no effect on the reaction performance (the reaction system and reagents are the same as those in the above embodiment 1). Figure 8 .

[0137] Example 4: Color development by mixing cresol red with methylene blue, bromocresol green, crystal violet and fast green

[0138] Cresol red was mixed with methylene blue, bromocresol green, crystal violet, and fast green for color development, and the LAMP detection method was the same as before. The results are shown in Table 7. Figure 9-12 .

[0139] Table 7. Color of double dye combinations at different ratios

[0140]

[0141]

[0142] Therefore, cresol red is mixed with methylene blue, bromocresol green, crystal violet and fast green to develop colors, and good distinction can be achieved in appropriate proportions.

[0143] Example 5: Color development by mixing hydroxynaphthol blue with phenol red and cresol red

[0144] The mixture of hydroxynaphthol blue and phenol red and cresol red was used for color development, and the LAMP detection method was the same as before. The color development is shown in Table 8 and Fig.13 , Fig.14 .

[0145] Table 8: Colors of different ratios of double dye combinations

[0146]

[0147] Therefore, mixing hydroxynaphthol blue with phenol red or cresol red can achieve good differentiation at the appropriate ratio.

[0148] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A LAMP visualization detection method, comprising using a visualization color reagent set in a LAMP reaction system; the dual display reagent comprises a color reagent 1 and a color reagent 2; The color developing reagent 1 changes from red to yellow in the range of 8.4±0.4 to 6.8±0.2; The color developing reagent 2 includes a blue, green or purple color developing reagent, which increases the color difference of the color developing reagent 1 and enhances the visual analysis of the LAMP reaction product.

2. The method according to claim 1, characterized in that The color developing reagent 1 includes: phenol red or cresol red, or a combination thereof; the color developing reagent 2 includes: methylene blue, bromocresol green, crystal violet, fast green or hydroxynaphthol blue, or a combination thereof; Preferably, the visualization color developing reagent group includes: phenol red and methylene blue, phenol red and bromocresol green, phenol red and crystal violet, phenol red and fast green, phenol red and hydroxynaphthol blue; cresol red and methylene blue, cresol red and bromocresol green, cresol red and crystal violet, cresol red and fast green, cresol red and hydroxynaphthol blue.

3. The method according to claim 1, characterized in that In the visualization color development reagent set: Phenol red:methylene blue is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Phenol red: bromocresol green is 1:1 to 4:1, preferably 1.5:1 to 3.5:1, more preferably 1.5:1 to 3:1; Phenol red: crystal violet is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Phenol red:fast green is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Phenol red: hydroxynaphthol blue is 0.5:1 to 3:1, preferably 1:1 to 2.5:1, more preferably 1:1 to 2:1; Cresol red:methylene blue is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Cresol red: bromocresol green is 0.5:1 to 4:1, preferably 1:1 to 3.5:1, more preferably 1.5:1 to 3:1; Cresol red: crystal violet is 1.5:1-4:1, preferably 1.5:1-3:1, more preferably 2:1-3:1; Cresol red:fast green is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; The ratio of cresol red to hydroxynaphthol blue is 0.5:1 to 3:1, more preferably 1:1 to 2.5:1, and more preferably 1:1 to 2:

1.

4. The method according to any one of claims 1 to 3, characterized in that: When performing the LAMP reaction, a sample to be tested containing a nucleic acid template is added to the LAMP reaction system; if the nucleic acid template contains a target nucleic acid that can be recognized and amplified by the primer, the color of the visualized color development reagent set changes; Preferably, the final concentration of the color developing reagent 1 or the color developing reagent 2 in the LAMP reaction system is 50 to 200 μM; preferably 100 to 180 μM; Preferably, the LAMP reaction system comprises: a visualization color development reagent set, dNTPs, DNA polymerase, MgSO4, and a reaction buffer; and optionally also comprises a LAMP primer set.

5. The method according to claim 4, characterized in that The visualization color developing reagent set is phenol red and methylene blue, and the color changes include: purple→green, blue→green, or purple→pink; or The visualization color developing reagent set is phenol red and bromocresol green, and the color changes include: purple→green, blue→green, purple→green, or pink→orange; or The visualization color reagent set is phenol red and crystal violet, and the color change includes: purple-red → brown; or The visualization color developing reagent set is phenol red and fast green, and the color change includes: red→green or orange→yellow; or The visualization color developing reagent set is phenol red and hydroxynaphthol blue, and the color changes include: blue→yellow, purple→green, purple→green, rose red→brown, pink→brown; or The visualization color developing reagent set is cresol red and methylene blue, and the color changes include: blue→green, purple→green, or reddish brown→green; or The visualization color developing reagent set is cresol red and bromocresol green, and the color changes include: blue→green, purple→green, purple→yellow-green, or pink→orange; or The visualization color developing reagent set is cresol red and crystal violet, and the color changes include: purple red→reddish brown, or purple red→brown; or The visualization color developing reagent set is cresol red and fast green, and the color changes include: pink→green, pink→green, purple→green, pink→yellow, or rose→orange; or The visualization color developing reagent set is cresol red and hydroxynaphthol blue, and the color changes include: blue→green, purple→green, brown→yellow, orange→yellow.

6. LAMP visualization detection reagent, the reagent is a visualization color reagent set, including color reagent 1 and color reagent 2; The color reagent 1 changes from red to yellow at 8.4±0.4; The color developing reagent 2 includes a blue, green or purple color developing reagent, which increases the color difference of the color developing reagent 1 and enhances the visual analysis of the LAMP reaction product.

7. The LAMP visualization detection reagent according to claim 6, characterized in that: The color developing reagent 1 includes: phenol red or cresol red, or a combination thereof; the color developing reagent 2 includes: methylene blue, bromocresol green, crystal violet, fast green or hydroxynaphthol blue, or a combination thereof; Preferably, the visualization color developing reagent group includes: phenol red and methylene blue, phenol red and bromocresol green, phenol red and crystal violet, phenol red and fast green, phenol red and hydroxynaphthol blue; cresol red and methylene blue, cresol red and bromocresol green, cresol red and crystal violet, cresol red and fast green, cresol red and hydroxynaphthol blue; More preferably, the visualization color developing reagent set comprises: Phenol red:methylene blue 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Phenol red: bromocresol green 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 1.5:1-3:1; Phenol red: crystal violet is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Phenol red:fast green is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Phenol red: hydroxynaphthol blue is 0.5:1 to 3:1, preferably 1:1 to 2.5:1, more preferably 1:1 to 2:1; Cresol red:methylene blue is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; Cresol red: bromocresol green is 0.5:1 to 4:1, preferably 1:1 to 3.5:1, more preferably 1.5:1 to 3:1; Cresol red: crystal violet is 1.5:1-4:1, preferably 1.5:1-3:1, more preferably 2:1-3:1; Cresol red:fast green is 1:1-4:1, preferably 1.5:1-3.5:1, more preferably 2:1-3:1; The ratio of cresol red to hydroxynaphthol blue is 0.5:1 to 3:1, more preferably 1:1 to 2.5:1, and more preferably 1:1 to 2:

1.

8. A reaction system for LAMP visualization detection, comprising the LAMP visualization detection reagent according to any one of claims 6 or 7; Preferably, the reaction system further comprises: Visualization reagent set, dNTPs, DNA polymerase, MgSO4, reaction buffer; optionally also includes LAMP primer set.

9. Use of the LAMP visualization detection reagent according to any one of claims 6 to 7, or the reaction system according to claim 7, for performing LAMP visualization detection, or for preparing a detection system or kit for performing LAMP visualization detection.

10. A LAMP visualization detection kit, comprising: The LAMP visualization detection reagent according to any one of claims 6 to 7; or the reaction system for LAMP visualization detection according to claim 8; preferably, the detection reagent or reaction system comprises a liquid system or a solid system.

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