A DNA detection method based on LAMP and immunochromatography
By combining LAMP amplification and immunochromatography, and utilizing FITC-labeled specific primers and colloidal gold to achieve visual detection, this method solves the problems of complex operation and equipment dependence in existing DNA detection methods, and achieves rapid detection with high sensitivity and specificity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG QINGLI BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-16
- Publication Date
- 2026-06-09
AI Technical Summary
Existing DNA testing methods are insufficient in terms of sensitivity and specificity to meet the needs of rapid on-site testing, and are complex to operate, requiring expensive instruments and equipment as well as professional technicians.
Combining LAMP amplification technology with immunochromatography, isothermal amplification was performed using FITC-labeled specific primers, and visual detection was achieved using colloidal gold. The results were read out using a double-antibody sandwich immunochromatography method.
It achieves highly sensitive, highly specific, and rapidly visualized DNA detection, simplifies operation, and is suitable for rapid on-site testing without the need for complex equipment or specialized technology.
Smart Images
Figure CN122168731A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical detection technology, specifically a DNA detection method based on LAMP and immunochromatography. Background Technology
[0002] In biomedical testing, efficient, sensitive, and rapid detection of target DNA has always been a key research focus and challenge. However, traditional DNA detection methods, such as polymerase chain reaction (PCR), while possessing high sensitivity and specificity, require complex thermal cycling equipment to control the reaction temperature for DNA denaturation, annealing, and extension steps. This not only increases testing costs but also limits its application in rapid on-site testing and resource-constrained environments.
[0003] Furthermore, while some other detection methods attempt to simplify procedures, they often fall short of ideal levels in terms of sensitivity and specificity. For example, some mainstream antibody-based detection methods, although simple to operate, have limited ability to detect low concentrations of target DNA, are prone to false negatives, and require expensive equipment and specialized technicians for operation and result analysis. This severely limits their widespread application in large-scale screening and primary healthcare units.
[0004] With the continuous development of biotechnology, isothermal amplification technology has gradually attracted attention. Among them, loop-mediated isothermal amplification (LAMP) technology is highly favored because it can rapidly and efficiently amplify target DNA at a constant temperature. However, LAMP technology usually requires subsequent detection methods such as gel electrophoresis to analyze the amplified products. These methods are cumbersome to operate and cannot achieve visual detection, which is not conducive to rapid on-site interpretation of results. Summary of the Invention
[0005] The purpose of this invention is to provide a DNA detection method based on LAMP and immunochromatography to solve the problems mentioned in the background art.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a DNA detection method based on LAMP and immunochromatography, comprising the following steps: S1. LAMP amplification and product labeling: Add the sample dilution to a LAMP reaction tube pre-filled with LAMP reaction solution, and use FITC (fluorescein) labeled specific primers to perform LAMP isothermal (65℃) amplification on the target DNA in the sample. S2. Immunochromatographic reagent detection: Using a double-antibody sandwich immunochromatographic method, colloidal gold is used to visualize and detect LAMP products labeled with FITC. S3. Result Determination: Positive result interpretation: The presence of red bands on both the test line (T line) and the control line (C line) indicates the presence of target DNA in the sample; Negative result interpretation: If only the control line (C line) shows a red band and the test line (T line) shows no band, it indicates that there is no target DNA in the sample; Invalid result determination: If there is no band on the control line (C line), it indicates that the test strip is invalid or the operation is incorrect, and the test needs to be repeated.
[0007] Furthermore, the LAMP amplification reaction system in step S1 includes: sample dilution solution (sterilized physiological saline or TE buffer, used to adjust the sample concentration to a suitable range for LAMP amplification), FITC-labeled specific primers, LAMP reaction buffer, Bst DNA polymerase, and a mixture of dNTPs. The reaction conditions are constant temperature incubation at 65°C for 30-60 minutes to achieve efficient amplification of target DNA.
[0008] Furthermore, the LAMP isothermal amplification involves primers specifically binding to the target DNA sequence to initiate a highly efficient amplification reaction, generating a large number of new DNA strands with FITC tags. The amplification reaction continues until the preset number of cycles or time is reached, ensuring that the target DNA is fully amplified. After amplification, each newly synthesized DNA strand carries a FITC tag.
[0009] Furthermore, the immunochromatographic reagent detection application uses an immunochromatographic reagent strip, and the immunochromatographic reagent strip includes a sample well, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. The preparation details are as follows: Gold particles labeled with anti-FITC antibody were coated onto the binding pad as signal carriers; Another anti-FITC antibody with different epitopes was coated on the detection line (T line) of the nitrocellulose membrane to capture the amplification product with FITC label; Goat anti-mouse IgG and goat anti-chicken IgY antibodies were coated on the control line (C line) of the nitrocellulose membrane to confirm that the test strip chromatography function was normal.
[0010] Furthermore, the diameter of the gold particles ranges from 20 nm to 40 nm to ensure good signal strength and stability.
[0011] Further, the specific operation of step S2 is as follows: the dilution of the LAMP reaction amplification product is added dropwise to the sample well of the test strip, so that the LAMP product with FITC labeling specifically binds to the gold particles on the binding pad to form a "gold particle-anti-FITC antibody-FITC labeled DNA product" complex. The complex is chromatographically precipitated upward along the nitrocellulose membrane under capillary action and captured by the anti-FITC antibody on the detection line (T line) to form a red detection band, indicating the presence of target DNA in the sample. Regardless of whether the sample contains the target, the unbound gold particles continue to chromatographically precipitate to the control line (C line) to form a second red band, which is used to confirm that the test strip chromatography function is normal.
[0012] This invention provides a DNA detection method based on LAMP and immunochromatography, which has the following beneficial effects: This invention combines LAMP technology with immunochromatography to form a dual detection strategy based on LAMP and immunochromatography. It can achieve efficient amplification of target DNA using LAMP primers under isothermal conditions, and label the amplification products by pre-incorporating FITC-labeled specific primers. Subsequently, the signal is visualized and read out using a double antibody sandwich immunochromatography method with colloidal gold. This achieves highly sensitive, highly specific, rapid, and visual detection of target DNA without the need for complex instruments and equipment. It is simple to operate, fast, and can meet the needs of rapid on-site detection. Attached Figure Description
[0013] Figure 1 This is a schematic diagram of the steps of a DNA detection method based on LAMP and immunochromatography according to the present invention. Figure 2 This is a schematic diagram of a DNA detection method based on LAMP and immunochromatography according to the present invention. Detailed Implementation
[0014] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.
[0015] Example 1: Detection of DNA from Specific Pathogens in Water Samples Step 1, Sample Collection and Processing: Collect 100 mL of water sample, concentrate the pathogen by centrifugation (10,000 rpm, 10 minutes), discard the supernatant, retain the precipitate, and then resuspend the precipitate in 1 mL of TE buffer (pH 8.0, containing 10 mM Tris-HCl and 1 mM EDTA) to obtain the sample dilution.
[0016] Step 2, LAMP amplification and product labeling: Add 25 μL of sample dilution to a LAMP reaction tube pre-filled with 75 μL of LAMP reaction solution. In this example, the LAMP reaction solution contains: 0.2 μM FITC-labeled specific primers (designed for specific gene sequences of pathogens), 1.4 mM dNTPs mixture, 8 U Bst DNA polymerase, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween 20, and 0.8 M betaine. Incubate the LAMP reaction tube at 65°C for 45 minutes for isothermal LAMP amplification.
[0017] Step 3: Immunochromatographic assay: Take 10 μL of the LAMP amplification product, dilute it with 90 μL of TE buffer, and then add the diluted LAMP product to the sample well of the immunochromatographic reagent strip. In this step, the immunochromatographic reagent strip, in addition to the sample well, includes a conjugate pad, a nitrocellulose membrane detection line (T line), and a nitrocellulose membrane control line (C line). The conjugate pad is coated with 30 nm gold particles labeled with anti-FITC antibody; the nitrocellulose membrane detection line (T line) is coated with another anti-FITC antibody with different epitopes; and the nitrocellulose membrane control line (C line) is coated with goat anti-mouse IgG and goat anti-chicken IgY antibodies. After adding the LAMP product, wait 5-10 minutes and observe the results.
[0018] Results: Both the test line (T line) and the control line (C line) showed red bands, indicating a positive result and the presence of specific pathogen DNA in the water sample.
[0019] Example 2: Detection of genetically modified DNA in food Step 1, Sample Collection and Processing: Take 10g of food sample (such as soy products), grind it into powder using a grinder, extract DNA with 50mL of physiological saline (0.9% NaCl), remove impurities by centrifugation (12,000rpm, 15 minutes), and retain the supernatant as sample dilution.
[0020] Step 2, LAMP amplification and product labeling: Add 50 μL of sample diluent to a LAMP reaction tube pre-loaded with 50 μL of LAMP reaction solution (the composition of the LAMP reaction solution is the same as in Example 1, but the specific primers are designed for the specific sequence of the transgenic component). Then, place the LAMP reaction tube in a constant temperature incubation at 65°C for 30 minutes to perform LAMP isothermal amplification.
[0021] Step 3, Immunochromatographic reagent detection: Take 20 μL of LAMP reaction amplification product, dilute it with 80 μL of physiological saline, and add the diluted LAMP product to the sample well of the immunochromatographic reagent strip. The preparation of the immunochromatographic reagent strip is the same as in Example 1. Wait 5-10 minutes and observe the results.
[0022] Results: Only the control line (C line) showed a red band, while the test line (T line) showed no band, indicating a negative result and meaning that there was no genetically modified DNA in the food.
[0023] Example 3: Rapid detection of viral DNA in clinical samples Step 1, Sample Collection and Processing: Collect clinical samples (pharyngeal swabs or blood), resuspend in 1 mL of physiological saline, remove cell debris by centrifugation (8,000 rpm, 5 minutes), and retain the supernatant as sample diluent.
[0024] Step 2, LAMP amplification and product labeling: Add 30 μL of sample dilution to a LAMP reaction tube pre-loaded with 70 μL of LAMP reaction solution (the composition of the LAMP reaction solution is the same as in Example 1, but the specific primers are designed for a specific sequence of viral DNA). Then, incubate the LAMP reaction tube at 65°C for 60 minutes to ensure that the viral DNA is fully amplified.
[0025] Step 3, Immunochromatographic reagent detection: Take 15 μL of LAMP reaction amplification product, dilute it with 85 μL of TE buffer, and add the diluted LAMP product to the sample well of the immunochromatographic reagent strip. The preparation of the immunochromatographic reagent strip is the same as in Example 1. Wait 5-10 minutes and observe the results.
[0026] Results: The control line (C line) showed no band, indicating an invalid test, possibly due to a faulty test strip or incorrect operation. After retesting, both the test line (T line) and the control line (C line) showed red bands, indicating a positive result and the presence of viral DNA in the clinical sample.
[0027] Example 4: Detection of DNA from bacteria associated with heavy metal pollution in environmental soil Step 1, Sample Collection and Processing: Collect approximately 20g of soil sample contaminated with heavy metals, place it in a sterile centrifuge tube, add 50mL of sterile physiological saline (0.9% NaCl), shake thoroughly to mix, let stand for 30 minutes to disperse bacterial cells, centrifuge at 10,000rpm for 10 minutes to remove soil particles and other impurities, and retain the supernatant as sample dilution.
[0028] Step 2, LAMP amplification and product labeling: Add 50 μL of sample diluent to a LAMP reaction tube pre-filled with 50 μL of LAMP reaction solution (the composition of the LAMP reaction solution is the same as in Example 1, but the specific primers are designed for specific gene sequences of bacteria related to heavy metal pollution). Then, place the LAMP reaction tube in a constant temperature of 65°C for 45 minutes to perform LAMP isothermal amplification.
[0029] Step 3, Immunochromatographic reagent detection: Take 20 μL of LAMP reaction amplification product, dilute it with 80 μL of TE buffer, and add the diluted LAMP product to the sample well of the immunochromatographic reagent strip. The preparation of the immunochromatographic reagent strip is the same as in Example 1. Wait 5-10 minutes and observe the results.
[0030] Results: Both the test line (T line) and the control line (C line) showed red bands, indicating a positive result, which means that the soil sample contained bacterial DNA related to heavy metal pollution.
[0031] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.
Claims
1. A DNA detection method based on LAMP and immunochromatography, characterized in that, Includes the following steps: S1. LAMP Amplification and Product Labeling: Add the sample dilution buffer to the LAMP reaction tube and use FITC-labeled specific primers to perform LAMP isothermal amplification on the target DNA in the sample. S2. Immunochromatographic reagent detection: Using a double-antibody sandwich immunochromatographic method, colloidal gold is used to visualize and detect LAMP products labeled with FITC. S3. Result Determination: Positive: Red bands appear on both the test line and the control line, indicating the presence of target DNA in the sample; Negative: Only the control line shows a red band, and the test line shows no band, indicating that there is no target DNA in the sample; Invalid: No stripe on the control line indicates that the test strip is invalid or the operation is incorrect, and it needs to be tested again.
2. The DNA detection method based on LAMP and immunochromatography according to claim 1, characterized in that, The sample diluent is TE buffer or physiological saline.
3. The DNA detection method based on LAMP and immunochromatography according to claim 1, characterized in that, The LAMP reaction solution contains FITC-labeled specific primers, dNTPs, Bst DNA polymerase, and a suitable buffer solution.
4. The DNA detection method based on LAMP and immunochromatography according to claim 1, characterized in that, The LAMP amplification was performed under isothermal conditions at 65°C, and the amplification reaction time was 30 to 60 minutes.
5. The DNA detection method based on LAMP and immunochromatography according to claim 4, characterized in that, The LAMP isothermal amplification involves primers specifically binding to the target DNA sequence, initiating a highly efficient amplification reaction, and generating a large number of new DNA strands with FITC tags.
6. The DNA detection method based on LAMP and immunochromatography according to claim 5, characterized in that, After the amplification is completed, each newly synthesized DNA strand carries a FITC tag.
7. The DNA detection method based on LAMP and immunochromatography according to claim 1, characterized in that, The immunochromatographic reagent detection application uses immunochromatographic reagent strips, which include a sample well, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. Preparation details are as follows: Gold particles labeled with anti-FITC antibody were coated onto the binding pad as signal carriers; Another anti-FITC antibody with different epitopes is coated on the detection line of the nitrocellulose membrane to capture amplification products with FITC labels; Goat anti-mouse IgG and goat anti-chicken IgY antibodies were coated on the control line of the nitrocellulose membrane to confirm that the test strip chromatography function was normal.
8. The DNA detection method based on LAMP and immunochromatography according to claim 7, characterized in that, The diameter of the gold particles ranges from 20 nm to 40 nm.
9. The DNA detection method based on LAMP and immunochromatography according to claim 7, characterized in that, The specific operation of step S2 is as follows: The dilution of the LAMP reaction amplification product is added dropwise to the sample well of the test strip, so that the LAMP product with FITC labeling specifically binds to the gold particles on the binding pad to form a complex. The complex is chromatographically precipitated upward along the nitrocellulose membrane under capillary action and captured by the anti-FITC antibody on the detection line, forming a red detection band, indicating the presence of target DNA in the sample; the unbound gold particles continue to chromatographically precipitate to the control line, forming a second red band, which is used to confirm that the test strip chromatography function is normal.
10. A DNA detection method based on LAMP and immunochromatography according to claim 9, characterized in that, The molecular structure of the complex is specifically: gold particles - anti-FITC antibody - FITC-labeled DNA product.