Colorful LAMP (loop-mediated isothermal amplification) rapid detection method and detection kit for nocardiasis of channa argus

Abstract

The invention discloses a coloration LAMP (loop-mediated isothermal amplification) rapid detection method for nocardiasis of snakehead. The method comprises the following steps: (1) extracting a to-be-detected snakehead tissue DNA (deoxyribonucleic acid) sample; (2) adding the DNA sample into an LAMP reaction system, wherein the reaction system comprises a specific primer group, Bst DNA polymerase, dNTPs, a reaction buffer solution and a color indicator hydroxycalcein, wherein the specific primer group is designed aiming at a nocardia argus 16S rRNA gene conserved region; (3) reacting for 20 minutes at the constant temperature of 65 DEG C in 25 microliters of sample adding system; and (4) after the reaction is finished, if nocardia argus exists in the sample, the reaction liquid becomes green, and if nocardia argus does not exist, the reaction liquid becomes orange. The method is simple and convenient to operate and suitable for convenient and rapid detection in production. The whole LAMP detection time is shortened to 30 minutes, and the detection can be completed; compared with a commercially available kit, the speed is increased by 41.59%; the primers are high in specificity, the sensitivity of the primers is 10-100 times higher than that of conventional PCR, low-concentration pathogenic bacteria can be effectively detected, and the primers are suitable for on-site rapid detection and large-scale screening in nocardiasis of channa argus and fry production.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic disease detection, and in particular to a chromogenic LAMP rapid detection method and a detection kit for Snakehead fish Nocardia infection. Background Art

[0002] Nocardiosis in snakehead fish (Canna argus) is a common aquatic disease that causes significant economic losses to the snakehead fish aquaculture industry. Currently, methods for detecting Nocardia in snakehead fish primarily include traditional culture, conventional PCR, fluorescent quantitative PCR, and commercially available detection kits. However, these methods have significant drawbacks: Traditional culture requires pathogen isolation, purification, and biochemical identification, taking up to 2-3 days and exhibiting low sensitivity, making it difficult to meet the demand for rapid detection; conventional PCR requires subsequent steps such as agarose gel electrophoresis, which is cumbersome and typically takes only 1-2 hours to detect; and its sensitivity is limited, making it difficult to detect low concentrations of pathogens; while fluorescent quantitative PCR, while highly sensitive, relies on expensive fluorescent quantitative PCR instrumentation, resulting in high testing costs and unsuitable for field-based aquaculture or large-scale screening; and commercially available detection kits typically take over 50 minutes, resulting in low efficiency, and some kits lack specificity, prone to false-positive results.

[0003] Therefore, developing a rapid, simple and sensitive method for detecting nocardiosis in Snakehead fish has important practical significance. Summary of the Invention

[0004] The present invention aims to address the deficiencies in the prior art and to provide a chromogenic LAMP rapid detection method and a detection kit for nocardia in snakehead fish that are simple to operate, rapid and efficient, highly sensitive, and suitable for on-site detection.

[0005] In order to achieve the above-mentioned purpose, the technical solution to be provided by the present invention is as follows:

[0006] A chromogenic LAMP rapid detection method for nocardia in snakehead fish comprises the following steps:

[0007] (1) extracting DNA samples from the snakehead fish tissue to be tested;

[0008] (2) adding the DNA sample to a LAMP reaction system, wherein the reaction system comprises a specific primer set designed for the conserved region of the 16S rRNA gene of Nocardia argus, BstDNA polymerase, dNTPs, a reaction buffer, and a color indicator hydroxycalcein;

[0009] (3) In a 25 μl sample system, react at 65°C for 20 minutes;

[0010] (4) After the reaction is completed, if Nocardia arguta is present in the sample, the reaction solution turns green; if Nocardia arguta is absent, the reaction solution turns orange.

[0011] Furthermore, the specific primer set includes: inner primers ChaFIP and ChaBIP; outer primers ChaF3 and ChaB3; wherein the sequences of the primers are as follows:

[0012] ChaF3: 5'-CCGCGGTAATACGTAGGGT-3' (SEQ ID NO: 1);

[0013] ChaB3: 5'-AGTTACTTCCCAGAGACCG-3' (SEQ ID NO: 2);

[0014] ChaFIP: 5'-TGTGAGTTTTCACGACCGACGTCCGGAATKACTGGGCGTA-3' (SEQ ID NO: 3);

[0015] ChaBIP: 5'-CAGGGGAGACTGGAATTCCTGGACCGGTGTTCCTCCTGAT-3' (SEQ ID NO: 4).

[0016] Furthermore, the total volume of the LAMP reaction system is 25ul, which contains: 1.6μM inner primers ChaFIP and ChaBIP; 0.2μM outer primers ChaF3 and ChaB3; 1.4mM dNTPs; 8U Bst DNA polymerase; 1× reaction buffer; and 20μM hydroxycalcein.

[0017] A kit for rapid detection of nocardia infection in snakehead fish using a chromogenic LAMP assay, comprising:

[0018] A specific primer set is designed for the conserved region of the 16S rRNA gene of Nocardia argus. The primer set comprises inner primers ChaFIP and ChaBIP, outer primers ChaF3 and ChaB3, Bst DNA polymerase, dNTPs, reaction buffer, and a color indicator calcein.

[0019] Furthermore, the invention also includes reagents for extracting DNA samples from snakehead fish tissues.

[0020] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0021] (1) The present invention does not require expensive equipment such as a fluorescent quantitative PCR instrument. It only requires a constant temperature metal bath to complete the amplification reaction, and the result can be determined by observing the color change with the naked eye, eliminating tedious steps such as agarose gel electrophoresis. It is suitable for rapid detection at grassroots breeding sites or in seedling production.

[0022] (2) By optimizing the reaction system, the present invention shortens the LAMP isothermal amplification time to 20 minutes, and the overall detection time (including reaction system configuration) is only 30 minutes. Compared with the commercially available ordinary test kit (average detection time of 51.3 minutes), the speed is increased by 41.59%, greatly improving the detection efficiency.

[0023] (3) The primer set designed by the present invention targets the conserved region of the 16S rRNA gene of Nocardia argus, has no cross-reaction with other common aquatic pathogens (such as Aeromonas hydrophila, Edwardsiella, etc.), and has strong specificity; and the detection sensitivity is 10-100 times higher than that of conventional PCR, and can effectively detect low-concentration pathogens (the minimum detection limit can reach 10 2 CFU / mL), which is suitable for the detection of trace pathogens in the early stages of the disease and large-scale screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the gel electrophoresis diagram of LAMP isothermal amplification of Nocardia argus (Note: M is Marker).

[0025] Figure 2 This is the LAMP color image of Nocardia argus after 20 minutes of isothermal amplification (Note: NG negative is orange; PG positive is green). DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the embodiments.

[0027] Example 1 Detection of Nocardia argus

[0028] (1) Extraction of DNA samples from the snakehead fish tissue to be tested:

[0029] (2) adding the DNA sample to a LAMP reaction system, which comprises a specific primer set designed for the conserved region of the 16S rRNA gene of Nocardia arguta, Bst DNA polymerase, dNTPs, a reaction buffer, and a color indicator hydroxycalcein;

[0030] The specific primer set includes: inner primers ChaFIP and ChaBIP; outer primers ChaF3 and ChaB3; the sequences of each primer are as follows:

[0031] ChaF3: 5'-CCGCGGTAATACGTAGGGT-3' (SEQ ID NO: 1);

[0032] ChaB3: 5'-AGTTACTTCCCAGAGACCG-3' (SEQ ID NO: 2);

[0033] ChaFIP: 5'-TGTGAGTTTTCACGACCGACGTCCGGAATKACTGGGCGTA-3' (SEQ ID NO: 3);

[0034] ChaBIP: 5'-CAGGGGAGACTGGAATTCCTGGACCGGTGTTCCTCCTGAT-3' (SEQ ID NO: 4);

[0035] (3) In a 25 μl sample loading system, react at a constant temperature of 65°C for 20 minutes; wherein the 25 μl system includes:

[0036] 1.6 μM of the internal primers ChaFIP and ChaBIP;

[0037] 0.2 μM outer primers ChaF3 and ChaB3;

[0038] 1.4 mM dNTPs;

[0039] 8 U of Bst DNA polymerase;

[0040] 1× reaction buffer;

[0041] 20 μM Calcein.

[0042] (4) After the reaction is completed, the sample is taken out. If Nocardia arguta is present in the sample, the reaction solution turns green. If Nocardia arguta is not present, the reaction solution turns orange.

[0043] Example 2 Specificity test

[0044] Nocardia arguta (positive control), Aeromonas hydrophila, Edwardsiella, Flavobacterium columnaris (negative control) and sterile water (blank control) were selected, and DNA was extracted according to the method of Example 1 and LAMP detection was performed.

[0045] The results showed that only the reaction solution of the Nocardia argus sample was green, while the reaction solutions of the other samples were orange, indicating that the primer set of the present invention had high specificity and no cross-reaction.

[0046] Example 3 Sensitivity test

[0047] The Nocardia arguta bacterial solution was diluted 10-fold (10 8 -10 1 CFU / mL), DNA was extracted from bacterial solutions of various dilutions, and the method of the present invention and conventional PCR were used for detection, respectively.

[0048] The results showed that the method of the present invention can detect 10 2CFU / mL of bacterial solution, while conventional PCR can only detect 10 3 -10 4 CFU / mL, indicating that the sensitivity of the present invention is 10-100 times higher than that of conventional PCR.

[0049] Example 4: Kit for rapid chromogenic LAMP detection of nocardia in snakehead fish

[0050] The kit includes: a specific primer set designed for the conserved region of the 16S rRNA gene of Nocardia argus (the same as in Example 1); Bst DNA polymerase; dNTPs; a reaction buffer; a color indicator hydroxycalcein (Calcein); and diatomaceous earth as a reagent for extracting DNA samples from Channa argus tissue.

[0051] The nucleic acid extraction method uses chitosan-modified diatomaceous earth to quickly extract nucleic acids from snakehead fish tissues. It innovatively uses chitosan-modified natural diatomaceous earth to quickly extract tissue nucleic acids. For the first time, the nucleic acid extraction from snakehead fish tissues is quickly completed through the three steps of release-enrichment-dissociation. This step is completed within 3 minutes.

[0052] The specific steps for extracting nucleic acids using the diatomaceous earth method are as follows:

[0053] (1) Mince aquatic animal tissue (e.g., liver), add 0.5 mL of water to a 1.5 mL centrifuge tube, and grind it with a homogenizer; centrifuge at 1000 r for 1 minute.

[0054] (2) In a 1.5 ml homogenate tube, use a pipette to draw 150 μl of the supernatant into a diatomaceous earth Ep tube;

[0055] (3) After the diatomaceous earth tube is shaken for a few seconds, it is placed in a metal bath at 95 degrees for 3 minutes. After the end, the precipitate is the extracted tissue nucleic acid. 2 μl of the precipitate is used as a tissue template for the next step of detection.

[0056] The detection method of the kit is the same as that of Example 1.

[0057] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A chromogenic LAMP rapid detection method for nocardia in snakehead fish, characterized in that: The following steps are involved: (1) extracting DNA samples from the snakehead fish tissue to be tested; (2) adding the DNA sample to a LAMP reaction system, wherein the reaction system comprises a specific primer set designed for the conserved region of the 16S rRNA gene of Nocardia arguta, Bst DNA polymerase, dNTPs, a reaction buffer, and a color indicator hydroxycalcein; (3) In a 25 μl sample system, react at 65°C for 20 minutes; (4) After the reaction is completed, if Nocardia arguta is present in the sample, the reaction solution turns green; if Nocardia arguta is absent, the reaction solution turns orange.

2. The detection method according to claim 1, wherein The specific primer set includes: inner primers ChaFIP and ChaBIP; outer primers ChaF3 and ChaB3; wherein the sequences of the primers are as follows: ChaF3: 5'-CCGCGGTAATACGTAGGGT-3' (SEQ ID NO: 1); ChaB3: 5'-AGTTACTTCCCAGAGACCG-3' (SEQ ID NO: 2); ChaFIP: 5'-TGTGAGTTTTCACGACCGACGTCCGGAATKACTGGGCGTA-3' (SEQ ID NO: 3); ChaBIP: 5'-CAGGGGAGACTGGAATTCCTGGACCGGTGTTCCTCCTGAT-3' (SEQ ID NO: 4).

3. The detection method according to claim 1, wherein The total volume of the LAMP reaction system is 25ul, which contains: 1.6 μM of the internal primers ChaFIP and ChaBIP; 0.2 μM outer primers ChaF3 and ChaB3; 1.4 mM dNTPs; 8 U of Bst DNA polymerase; 1× reaction buffer; 20 μM Calcein.

4. A kit for rapid detection of nocardiosis in snakehead fish using a chromogenic LAMP assay, characterized in that: include: A specific primer set designed for the conserved region of the 16S rRNA gene of Nocardia argus, comprising inner primers ChaFIP and ChaBIP, and outer primers ChaF3 and ChaB3; Bst DNA polymerase; dNTPs; Reaction buffer; Color indicator: Calcein.

5. The kit according to claim 4, characterized in that The sequences of the specific primer sets are as follows: ChaF3: 5'-CCGCGGTAATACGTAGGGT-3' (SEQ ID NO: 1); ChaB3: 5'-AGTTACTTCCCAGAGACCG-3' (SEQ ID NO: 2); ChaFIP: 5'-TGTGAGTTTTCACGACCGACGTCCGGAATKACTGGGCGTA-3' (SEQ ID NO: 3); ChaBIP: 5'-CAGGGGAGACTGGAATTCCTGGACCGGTGTTCCTCCTGAT-3' (SEQ ID NO: 4).

6. The kit according to claim 4, wherein Also included are reagents for extracting DNA samples from snakehead fish tissue.

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