LAMP (loop-mediated isothermal amplification) double-signal visual detection indicator based on SYTO 9 and application of LAMP double-signal visual detection indicator

Abstract

The invention relates to the technical field of molecular biology, in particular to an LAMP (Loop-Mediated Isothermal Amplification) double-signal visual detection indicator based on SYTO 9 and application of the LAMP double-signal visual detection indicator. SYTO 9 is adopted as a fluorescent indicator, a phenol red and hydroxynaphthol blue mixture is adopted as a color indicator, and it is found that after SYTO 9 is added, the visual sensitivity is increased from 20 cfu / reaction to 2 cfu / reaction; by means of the method, the low copy number can be detected, the false positive rate is reduced, and the problem that weak positive caused by too low template concentration is difficult to distinguish during photographing retention or visual distinguishing can be perfectly solved. The color indicator provided by the invention can simultaneously use a direct visual method and a real-time fluorescence amplification method, has the characteristics of high resolution, high sensitivity and low false positive rate, realizes the effect of judging whether a sample is positive or negative by naked eyes in combination with double signals of a fluorescence amplification curve, and is high in judgment result accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a SYTO 9-based LAMP dual-signal visual detection indicator and applications thereof. Background Art

[0002] Loop-mediated isothermal amplification (LAMP) is a nucleic acid isothermal amplification technique first proposed by Notomi in 2000. Its principle is to design 4 to 6 specific primers for 6 to 8 target regions, and then rapidly amplify the target under the action of Bst DNA polymerase with strand displacement activity. Since its publication, this technology has attracted widespread attention and research. With its low requirements for reaction equipment, strong specificity, and high sensitivity, it has largely overcome the shortcomings of variable temperature detection technology and better meets the needs of rapid on-site testing.

[0003] The LAMP reaction is very fast and efficient, and can produce and accumulate a large amount of amplification products in a short time. The detection of LAMP products is usually divided into two forms. The first is the detection of double strands of the main product. The detection of double strands is usually carried out using agarose electrophoresis gel technology, or by adding nucleic acid dyes after the reaction to observe the fluorescence color difference to read; the second is to use the by-products to detect and judge the results. In the LAMP amplification process, in addition to the production of double strands, there is also a process in which dNTP drops pyrophosphate as the reaction proceeds. Pyrophosphate exists in the solution in the form of pyrophosphate and hydrogen ions. Free magnesium ions encounter pyrophosphate to form magnesium pyrophosphate. Therefore, in the result judgment of by-products, the concentration can be indicated by color development using a pH indicator, the magnesium pyrophosphate precipitation can be measured by a turbidimeter, or the Mg can be measured by a metal indicator. 2+ On the basis of avoiding aerosol contamination caused by opening the lid and ensuring high readability of the results, achieving visual detection of the LAMP endpoint is an urgent problem to be solved.

[0004] In existing LAMP detection methods, single colorants (such as phenol red and hydroxy naphthol blue) have the following problems: the color change is not significant. For example, phenol red changes from pink to orange (dark → light) after the reaction, and HNB changes from violet to sky blue (dark → light). The color contrast is reduced, and weak positive samples are difficult to interpret with the naked eye. At the same time, the sensitivity is limited: a single dye relies on a single color development mechanism (such as pH or metal ion response), and it is impossible to improve sensitivity through the synergy of multiple signals.

[0005] Therefore, it is crucial to provide a technical solution that can solve the problems of the existing technology such as the difficulty in distinguishing weak positive samples and the high false positive rate. Summary of the Invention

[0006] To address the above-mentioned issues, the present invention aims to provide a SYTO 9-based LAMP dual-signal visual detection indicator and its application. By introducing the SYTO 9 fluorescent dye, the present invention significantly improves detection sensitivity and enables closed-tube dual-signal interpretation of results. By mixing phenol red with HNB, the reaction solution color changes from light purple (negative) to dark gray (positive) for the first time, changing from light to dark, significantly enhancing the color contrast for naked-eye interpretation. Furthermore, the present invention innovatively introduces SYTO 9, which can efficiently penetrate cell membranes and bind to nucleic acids, significantly enhancing the fluorescence signal. The SYTO 9 also synergizes with color-developing indicators (such as phenol red / HNB) to improve detection sensitivity to 2 cfu / reaction through dual-signal verification of color change and fluorescence signal, solving the problem of interpreting weakly positive samples.

[0007] During the LAMP amplification process, H + As a by-product, it causes the pH value of the reaction system to drop, and the color change area of ​​phenol red coincides with the appropriate pH range for LAMP amplification.

[0008] If a single dye (such as phenol red or HNB) is used as a color indicator, the color changes from dark to light before and after the reaction. The difference between a weak positive and a negative result is relatively subtle, making it impossible to quickly and accurately interpret the results, reducing the speed and accuracy of on-site diagnosis. Using phenol red as a pH indicator combined with a mixed dye solution of HNB for result judgment is more obvious than using a single dye. The reaction solution appears light purple in the negative state and turns dark gray in the positive state. This can achieve a change in solution color from light to dark before and after the reaction, making it more suitable for visual interpretation of reaction results.

[0009] SYTO 9 is a green fluorescent nucleic acid dye that can penetrate cell membranes and bind to DNA (and RNA) with high affinity. Once a positive reaction occurs in the detection system, a large amount of DNA product is produced. SYTO 9 will bind to DNA and show a significantly enhanced fluorescence signal. The maximum excitation and emission wavelengths are 483nm and 503nm, respectively. It can be detected through the FAM channel, and the fluorescence amplification curve can be observed on a fluorescence quantitative PCR instrument.

[0010] This dual-signal visualization detection method based on the LAMP system utilizes the fluorescent dye SYTO 9 and the color indicators phenol red and HNB, combining the color changes observed by the naked eye before and after the reaction with the fluorescence amplification curve detected by a fluorescence quantitative PCR instrument. This method allows positive samples to exhibit a significant color change (e.g., light purple to dark gray) using the color developer (e.g., phenol red / HNB) under natural light, while simultaneously displaying a specific fluorescence amplification curve on the fluorescence quantitative PCR instrument. These two signals mutually verify each other, avoiding errors in single-interpretation interpretation. Furthermore, SYTO 9 can be premixed directly into the reaction system, eliminating the need to open the lid to add dye, completely eliminating the risk of aerosol contamination.

[0011] The purpose of the present invention can be achieved by the following technical solutions:

[0012] The first object of the present invention is to provide a SYTO 9-based LAMP dual-signal visual detection indicator, comprising SYTO 9 and one or both of phenol red and HNB;

[0013] The concentration of SYTO 9 is 0.1× to 1×, the concentration of phenol red is 0.1 mM to 1 mM, and the concentration of HNB is 0.1 mM to 1 mM.

[0014] In one embodiment of the present invention, the concentration of SYTO 9 is 0.3× to 0.6×, the concentration of phenol red is 0.2 mM to 0.5 mM, and the concentration of HNB is 0.2 mM to 0.5 mM;

[0015] Preferably, the concentration of SYTO 9 is 0.3×, the concentration of phenol red is 0.35 mM, and the concentration of HNB is 0.35 mM.

[0016] In the present invention, phenol red is used as a pH indicator and SYTO 9 is used as a fluorescent indicator. Phenol red, HNB, and SYTO 9 cooperate with each other within a certain concentration range to detect low copy numbers, reduce false positive rates, and perfectly solve the problem of difficulty in distinguishing weak positives caused by too low template concentration during photographic retention or visual identification.

[0017] The second object of the present invention is to provide a use of a SYTO 9-based LAMP dual-signal visual detection indicator in the preparation of a LAMP detection system.

[0018] A third object of the present invention is to provide a LAMP detection system comprising Tris-HCl, betaine, dNTPs, magnesium sulfate, sodium chloride, ammonium sulfate, potassium hydroxide, Bst DNA polymerase, loop-mediated isothermal amplification primers, and the above-mentioned SYTO 9-based LAMP dual-signal visual detection indicator.

[0019] In one embodiment of the present invention, the loop-mediated isothermal amplification primers include FIP primer, BIP primer, F3 primer, B3 primer, LF primer and LB primer.

[0020] In one embodiment of the present invention, in the LAMP detection system, the concentration of Tris-HCl is 10mM to 30mM; the concentration of betaine is 0.25M to 1M; the concentration of dNTP is 1mM to 1.8mM; the concentration of magnesium sulfate solution is 3.75mM to 7.5mM; the concentration of sodium chloride solution is 10mM to 100mM; the concentration of ammonium sulfate solution is 5mM to 15mM; the concentration of potassium hydroxide is 1mM to 10mM; the concentration of Bst The concentration of DNA polymerase is 0.4U / μL~1.2U / μL; the concentration of the FIP primer is 0.8mM~3.2mM; the concentration of the BIP primer is 0.8mM~3.2mM; the concentration of the F3 primer is 0.1mM~0.4mM; the concentration of the B3 primer is 0.1mM~0.4mM; the concentration of the LF primer is 0.2mM~0.8mM; and the concentration of the LB primer is 0.2mM~0.8mM.

[0021] In one embodiment of the present invention, in the LAMP detection system, the concentration of Tris-HCl is 15 mM; the concentration of betaine is 0.8 M; the concentration of dNTP is 1.4 mM; the concentration of magnesium sulfate solution is 6.25 mM; the concentration of sodium chloride solution is 50 mM; the concentration of ammonium sulfate solution is 10 mM; the concentration of potassium hydroxide is 4 mM; the concentration of Bst DNA polymerase is 0.8 U / μL; the concentration of FIP primer is 1.6 mM; the concentration of BIP primer is 1.6 mM; the concentration of F3 primer is 0.2 mM; the concentration of B3 primer is 0.2 mM; the concentration of LF primer is 0.4 mM; and the concentration of LB primer is 0.4 mM.

[0022] The fourth object of the present invention is to provide an application of a LAMP detection system in LAMP detection.

[0023] A fifth object of the present invention is to provide a method for detecting LAMP products, comprising the following steps:

[0024] The LAMP detection system is mixed with the sample to be tested and then amplified at a constant temperature. When the reaction solution turns from light purple to dark gray, the sample to be tested is determined to be positive.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention uses SYTO 9 as a fluorescent indicator and a mixture of phenol red and hydroxynaphthol blue as a color indicator. Experiments show that after adding SYTO 9, the visual sensitivity is increased from 20 cfu / reaction to 2 cfu / reaction, which can detect low copy numbers, reduce false positive rates, and perfectly solve the problem of difficulty in distinguishing weak positives caused by low template concentration when photographing or visually distinguishing. The color indicator of the present invention can be used simultaneously with direct visual method and real-time fluorescence amplification method, and has the characteristics of high resolution, high sensitivity, and low false positive rate. It achieves the effect of judging whether a sample is positive or negative by visually combining the dual signal verification of the fluorescence amplification curve, and the judgment result has a high accuracy rate.

[0027] (2) The synergistic effect of SYTO 9 and the color developer (phenol red / HNB) is reflected in: the color contrast between negative and positive samples is enhanced, and the color developer assists in amplifying the color difference visible to the naked eye. Traditional single color developers (such as phenol red or HNB) change color slightly after the reaction (phenol red: pink → orange, HNB: violet → sky blue), and the color changes from dark to light, making weak positive samples difficult to distinguish with the naked eye; the mixed dye used in the present invention, through the synergistic effect of phenol red and HNB, changes the color of the reaction solution from light purple (negative) to dark gray (positive), and the color changes from light to dark, significantly improving the intuitiveness and reliability of naked eye interpretation.

[0028] (3) The present invention adopts a one-tube premix solution, which does not require the addition of additional enzymes and has the characteristics of simple operation and pollution control.

[0029] (4) Not only can the test results be quickly determined easily, but there is also no need to open the reaction tube after the reaction is completed, thus avoiding aerosol contamination.

[0030] In summary, the present invention achieves a simultaneous improvement in sensitivity, specificity, and operational convenience through the synergistic effect of the fluorescence enhancement properties of SYTO 9 and the phenol red / HNB mixed color development system, providing an innovative solution for the on-site and precise application of LAMP technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The following are the amplification curve results of the fluorescence quantitative PCR instrument with a constant temperature reaction at 64°C for 60 minutes; among them, A is the fluorescence amplification curve obtained at a Syto 9 concentration of 0.1×, B is the fluorescence amplification curve obtained at a Syto 9 concentration of 0.3×, C is the fluorescence amplification curve obtained at a Syto 9 concentration of 0.6×, and D is the fluorescence amplification curve obtained at a Syto 9 concentration of 1×. a indicates a Salmonella concentration of 200 cfu / reaction, b indicates a Salmonella concentration of 100 cfu / reaction, c indicates a Salmonella concentration of 20 cfu / reaction, d indicates a Salmonella concentration of 2 cfu / reaction, and e is a negative control (ddH2O).

[0032] Figure 2 The following are photos of the reaction tube results after reaction under natural light; among them, A is a dye solution containing only phenol red, B is a dye solution containing only HNB, C is a dye solution containing phenol red and HNB, and D is a dye solution containing phenol red, HNB, and SYTO 9. a indicates a Salmonella concentration of 400 cfu / reaction, b indicates a Salmonella concentration of 200 cfu / reaction, c indicates a Salmonella concentration of 100 cfu / reaction, d indicates a Salmonella concentration of 20 cfu / reaction, e indicates a Salmonella concentration of 2 cfu / reaction, and f is a negative control (ddH2O).

[0033] Figure 3 The following is a picture of the amplification curve results of the fluorescence quantitative PCR instrument with a constant temperature reaction at 64°C for 30 minutes; among them, a represents a Salmonella concentration of 400 cfu / reaction, b represents a Salmonella concentration of 200 cfu / reaction, c represents a Salmonella concentration of 100 cfu / reaction, d represents a Salmonella concentration of 20 cfu / reaction, e represents a Salmonella concentration of 2 cfu / reaction, and f represents a negative control (ddH2O).

[0034] Figure 4 The results of the reaction tubes after the reaction under natural light are shown in Figure 2. A is the reaction tube of the dual-signal visualization LAMP detection system before the reaction, B is the reaction tube of the dual-signal visualization LAMP detection system after the reaction, a is the dual-signal visualization LAMP detection reaction tube containing Salmonella, b is the dual-signal visualization LAMP detection reaction tube containing Staphylococcus aureus, c is the dual-signal visualization LAMP detection reaction tube containing Escherichia coli, d is the dual-signal visualization LAMP detection reaction tube containing Klebsiella pneumoniae, and e is the dual-signal visualization LAMP detection reaction tube containing Pseudomonas aeruginosa. Signal visualization LAMP detection reaction tube, f is a dual-signal visualization LAMP detection reaction tube containing Enterococcus faecalis, g is a dual-signal visualization LAMP detection reaction tube containing Enterococcus faecium, h is a dual-signal visualization LAMP detection reaction tube containing Proteus vulgaris, i is a dual-signal visualization LAMP detection reaction tube containing Vibrio parahaemolyticus, j is a dual-signal visualization LAMP detection reaction tube containing Stenotrophomonas maltophilia, k is a dual-signal visualization LAMP detection reaction tube containing Acinetobacter baumannii, and l is a negative control (ddH2O).

[0035] Figure 5 Figure 2 is the amplification curve result of the fluorescence quantitative PCR instrument with a constant temperature reaction at 64℃ for 30 minutes; among them, a is the reaction tube containing Salmonella enterica, b is the reaction tube containing Staphylococcus aureus, c is the reaction tube containing Escherichia coli, d is the reaction tube containing Klebsiella pneumoniae, e is the reaction tube containing Pseudomonas aeruginosa, f is the reaction tube containing Enterococcus faecalis, g is the reaction tube containing Enterococcus faecium, h is the reaction tube containing Proteus vulgaris, and l is the negative control (ddH2O). DETAILED DESCRIPTION

[0036] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] In the following examples, Salmonella is Salmonella typhimurium (ATCC 13311); Staphylococcus aureus was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of B81854 (A); Escherichia coli was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of BMZ064412; Klebsiella pneumoniae was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of BMZ134355; Pseudomonas aeruginosa was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of BMZ134736; Enterococcus faecalis was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with a product number of B94699; Enterococcus faecium was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. Co., Ltd., product number B00006; Proteus vulgaris was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number BMZ122565; Vibrio parahaemolyticus was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number B87538; Stenotrophomonas maltophilia was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number BMZ133852; Acinetobacter baumannii was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number B00001; unless otherwise specified, all reagents used were commercially available reagents, and the detection means and methods used were conventional detection means and methods in this field.

[0038] Example 1

[0039] This example provides Salmonella-based loop-mediated isothermal amplification primers.

[0040] Six corresponding LAMP primers (S-F3, S-B3, S-FIP, S-BIP, S-LF, and S-LB) were used according to the characteristic nucleic acid sequences of Salmonella; all primers were synthesized by Shanghai Diying Biotechnology Co., Ltd., and the primer sequences are shown in Table 1.

[0041] Table 1. Salmonella primer sequence list

[0042]

[0043] Example 2

[0044] This example provides a comparison of the amplification effects of various LAMP detection systems, as follows:

[0045] (1) Preparation of SYTO 9 premixes with different concentrations

[0046] In the premix, the fluorescent indicator SYTO 9 (diluted with 20× stock solution, Shanghai Fujin Technology Co., Ltd.) was set to four concentration gradients of 0.1×, 0.3×, 0.6×, and 1×, Tris-HCl 15mM, betaine 0.8M, dNTP 1.4mM, magnesium sulfate solution 6.25mM, sodium chloride solution 50mM, ammonium sulfate solution 10mM, potassium hydroxide solution 4mM, Bst DNA polymerase (purchased from Shanghai Fujin Technology Co., Ltd.) 0.8U / μL, S-FIP primer 1.6mM, S-BIP primer 1.6mM, S-F3 primer 0.2mM, S-B3 primer 0.2mM, S-LF primer 0.4mM, and S-LB primer 0.4mM.

[0047] (2) LAMP reaction

[0048] Each premixed solution in step (1) was added with different concentrations of Salmonella solution (concentrations were 200, 100, 20, and 2 cfu / reaction) to form a LAMP reaction solution. The reaction volume was 20 μL, and the reaction solution was placed at 64° C. for 60 minutes.

[0049] (3) Read the results

[0050] pass Figure 1 It can be found that when the Syto 9 concentration is 0.1×, the curve with a Salmonella concentration of 2 cfu / reaction shows an amplification reaction after 30 minutes; when the Syto 9 concentration is 0.3×, 0.6×, and 1×, the four concentration gradients of Salmonella show specific amplification within 30 minutes during the constant temperature amplification process, and the amplification effect is good.

[0051] Considering the impact of experimental costs, 0.3× was selected as the optimal Syto 9 concentration in subsequent experiments.

[0052] Example 3

[0053] This example provides the effects of different dye solutions on detection sensitivity, as follows:

[0054] (1) Preparation of different dye solution combinations

[0055] Group A (the dye solution contains only phenol red): phenol red 0.35 mM, Tris-HCl 15 mM, betaine 0.8 M, dNTP 1.4 mM, magnesium sulfate solution 6.25 mM, sodium chloride solution 50 mM, ammonium sulfate solution 10 mM, potassium hydroxide solution 4 mM, Bst DNA polymerase (purchased from Shanghai Fujin Technology Co., Ltd.) 0.8 U / μL, S-FIP primer 1.6 mM, S-BIP primer 1.6 mM, S-F3 primer 0.2 mM, S-B3 primer 0.2 mM, S-LF primer 0.4 mM, S-LB primer 0.4 mM.

[0056] Group B (staining solution contains only HNB): HNB 0.35mM, Tris-HCl 15mM, betaine 0.8M, dNTP 1.4mM, magnesium sulfate solution 6.25mM, sodium chloride solution 50mM, ammonium sulfate solution 10mM, potassium hydroxide solution 4mM, Bst DNA polymerase (purchased from Shanghai Fujin Technology Co., Ltd.) 0.8U / μL, S-FIP primer 1.6mM, S-BIP primer 1.6mM, S-F3 primer 0.2mM, S-B3 primer 0.2mM, S-LF primer 0.4mM, S-LB primer 0.4mM.

[0057] Group C (staining solution contains phenol red and HNB): phenol red 0.35 mM, HNB 0.35 mM, Tris-HCl 15 mM, betaine 0.8 M, dNTP 1.4 mM, magnesium sulfate solution 6.25 mM, sodium chloride solution 50 mM, ammonium sulfate solution 10 mM, potassium hydroxide solution 4 mM, BstDNA polymerase (purchased from Shanghai Fujin Technology Co., Ltd.) 0.8 U / μL, S-FIP primer 1.6 mM, S-BIP primer 1.6 mM, S-F3 primer 0.2 mM, S-B3 primer 0.2 mM, S-LF primer 0.4 mM, S-LB primer 0.4 mM.

[0058] Group D (staining solution contains phenol red, HNB, and SYTO 9): phenol red 0.35 mM, HNB 0.35 mM, SYTO 9 0.3× (referring to 20× SYTO 9 stock solution diluted to 0.3×, the same below), Tris-HCl 15 mM, betaine 0.8 M, dNTP 1.4 mM, magnesium sulfate solution 6.25 mM, sodium chloride solution 50 mM, ammonium sulfate solution 10 mM, potassium hydroxide solution 4 mM, Bst DNA polymerase (purchased from Shanghai Fujin Technology Co., Ltd.) 0.8 U / μL, S-FIP primer 1.6 mM, S-BIP primer 1.6 mM, S-F3 primer 0.2 mM, S-B3 primer 0.2 mM, S-LF primer 0.4 mM, and S-LB primer 0.4 mM.

[0059] (2) LAMP reaction

[0060] The premixed solutions of the above-mentioned different dye solution combinations (group A, group B, group C, and group D) were added to different concentration gradient Salmonella solutions (concentrations of 400, 200, 100, 20, and 2 cfu / reaction, respectively) to form LAMP reaction solutions. The reaction volume was 20 μL, and the reaction solution was placed at 64°C for 30 minutes.

[0061] (3) Read the results

[0062] Under natural light, observe the color change of the reaction solution with the naked eye. Figure 2 As shown, it shows that when the Salmonella concentrations a to e corresponding to the dual-signal visual dye indicator under natural light are approximately 400, 200, 100, 20, and 2 cfu / reaction, and f is the negative control, it can be seen that when the Salmonella concentrations corresponding to a to e in group D are, the reaction tube solution undergoes a significant color change from purple (negative) to gray (positive).

[0063] Accordingly, combined with the results of fluorescence quantitative PCR instrument, Figure 3 As shown, the fluorescence amplification curves corresponding to the Salmonella concentrations a through e appear. Clearly, the difference between negative and positive results using the dual-signal visualization indicator premix is ​​significant, enabling dual-signal visualization interpretation of results. Furthermore, the sensitivity of the LAMP dual-signal visualization system for detecting Salmonella is approximately 2 cfu / reaction. The synergistic effect of SYTO 9, phenol red, and HNB significantly enhances detection sensitivity, resolving the difficulty in distinguishing weak positives caused by low template concentrations during photographic retention or visual identification.

[0064] Example 4

[0065] This example provides a specificity test of a dual-signal visual LAMP detection system, as follows:

[0066] (1) Dual-signal visual indicator premix configuration

[0067] Dual-signal visual indicator premix: The optimized formula of Group D in Example 3 (phenol red 0.35 mM, HNB 0.35 mM, SYTO 9 0.3×) was used, and the other components were the same as in Example 1.

[0068] (2) LAMP reaction

[0069] The premixed solution containing the dual-signal visual color indicator was added to different species of experimental bacteria (Salmonella enterica, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, Proteus vulgaris, Vibrio parahaemolyticus, Stenotrophomonas maltophilia, and Acinetobacter baumannii) to form a LAMP reaction solution. The reaction volume was 20 μL, and the reaction solution was placed at 64°C for 30 minutes.

[0070] (3) Read the results

[0071] pass Figure 4 It can be found that the Salmonella strain reaction tube solution corresponding to a has a significant color change from purple (negative) to gray (positive), while the non-Salmonella strain reaction tube solutions corresponding to b to k and the negative control corresponding to l have no color change. Figure 5 As can be seen, the Salmonella reaction (a) exhibits a fluorescence amplification curve, while the non-Salmonella reaction solution (bk) and the negative control (l) show no nonspecific amplification. This demonstrates a clear distinction between positive and negative results with the dual-signal visual indicator premix, demonstrating good specificity.

[0072] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A SYTO 9-based LAMP dual-signal visual detection indicator, characterized in that: Includes SYTO 9, and one or both of phenol red and hydroxynaphthol blue; The concentration of SYTO 9 is 0.1× to 1×, the concentration of phenol red is 0.1 mM to 1 mM, and the concentration of hydroxynaphthol blue is 0.1 mM to 1 mM.

2. The SYTO 9-based LAMP dual-signal visual detection indicator according to claim 1, characterized in that: The concentration of SYTO 9 is 0.3× to 0.6×, the concentration of phenol red is 0.2 mM to 0.5 mM, and the concentration of hydroxynaphthol blue is 0.2 mM to 0.5 mM.

3. The SYTO 9-based LAMP dual-signal visual detection indicator according to claim 1, characterized in that: The concentration of SYTO 9 was 0.3×, the concentration of phenol red was 0.35 mM, and the concentration of hydroxynaphthol blue was 0.35 mM.

4. Use of the SYTO 9-based LAMP dual-signal visual detection indicator according to any one of claims 1 to 3 in preparing a LAMP detection system.

5. A LAMP detection system, characterized in that: The method comprises Tris-HCl, betaine, dNTP, magnesium sulfate, sodium chloride, ammonium sulfate, potassium hydroxide, Bst DNA polymerase, loop-mediated isothermal amplification primers and the SYTO9-based LAMP dual-signal visual detection indicator according to any one of claims 1 to 3.

6. A LAMP detection system according to claim 5, characterized in that, The loop-mediated isothermal amplification primers include FIP primer, BIP primer, F3 primer, B3 primer, LF primer and LB primer.

7. A LAMP detection system according to claim 6, characterized in that, In the LAMP detection system, the concentration of Tris-HCl is 10mM to 30mM; the concentration of betaine is 0.25M to 1M; the concentration of dNTP is 1mM to 1.8mM; the concentration of magnesium sulfate solution is 3.75mM to 7.5mM; the concentration of sodium chloride solution is 10mM to 100mM; the concentration of ammonium sulfate solution is 5mM to 15mM; the concentration of potassium hydroxide is 1mM to 10mM; the concentration of Bst The concentration of DNA polymerase is 0.4U / μL~1.2U / μL; the concentration of the FIP primer is 0.8mM~3.2mM; the concentration of the BIP primer is 0.8mM~3.2mM; the concentration of the F3 primer is 0.1mM~0.4mM; the concentration of the B3 primer is 0.1mM~0.4mM; the concentration of the LF primer is 0.2mM~0.8mM; and the concentration of the LB primer is 0.2mM~0.8mM.

8. A LAMP detection system according to claim 7, characterized in that, In the LAMP detection system, the concentration of Tris-HCl is 15 mM; the concentration of betaine is 0.8 M; the concentration of dNTP is 1.4 mM; the concentration of magnesium sulfate solution is 6.25 mM; the concentration of sodium chloride solution is 50 mM; the concentration of ammonium sulfate solution is 10 mM; the concentration of potassium hydroxide is 4 mM; the concentration of Bst DNA polymerase is 0.8 U / μL; the concentration of FIP primer is 1.6 mM; the concentration of BIP primer is 1.6 mM; the concentration of F3 primer is 0.2 mM; the concentration of B3 primer is 0.2 mM; the concentration of LF primer is 0.4 mM; and the concentration of LB primer is 0.4 mM.

9. Use of the LAMP detection system according to claim 5 in LAMP detection.

10. A method for detecting LAMP products, characterized in that: The following steps are involved: The LAMP detection system according to claim 5 is mixed with the sample to be tested and then amplified at a constant temperature. When the reaction solution changes from light purple to dark gray, the sample to be tested is determined to be positive.

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