A LAMP primer set and kit for detecting Vibrio harveyi

Abstract

This invention discloses a LAMP primer set and kit for detecting Vibrio harveyi. The invention provides a LAMP primer set for detecting Vibrio harveyi, and further provides a kit and method for detecting Vibrio harveyi. The LAMP primer set described in this invention has high detection specificity and high detection sensitivity, with a limit of detection of 2.4 × 10⁻⁶. 0 The copy / μL method for detecting Vibrio harveyi constructed using this method has the advantages of simple operation and accurate detection results. It overcomes the shortcomings of existing molecular biological detection methods for Vibrio harveyi, such as long detection time, low sensitivity, high cost, and difficulty in field application. It is suitable for rapid field detection in grassroots farms and helps in the prevention and control of vibriosis.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology. More specifically, it relates to a LAMP primer set and kit for detecting Vibrio harveyi. Background Technology

[0002] Vibrio harveyi ( Vibrio harveyi Vibrio harveyi is a curved, rod-shaped, luminescent, halophilic Gram-negative bacterium. With the rapid development of aquaculture, the increasing stocking density, and other factors, aquatic animal diseases frequently break out. Vibrio infection caused by Vibrio harveyi is one of the most serious, causing enormous harm to the aquaculture industry.

[0003] Vibrio harveyi, a key pathogen of vibriosis, can infect various aquatic animals, including marine fish, shrimp, and crustaceans, with a mortality rate of almost 100% in juvenile shrimp. Besides shrimp, Vibrio harveyi can also infect sea bass (…). Centrupomus undecimalis ), oblique grouper ( Epinephelus coioides ) and turbot ( Scophthalmus maximus When farmed fish, such as those infected with Vibrio harveyi, are infected, they will exhibit symptoms such as bulging eyes, congestion, or gastroenteritis, and in severe cases, death. Therefore, in order to effectively prevent vibriosis caused by Vibrio harveyi, it is essential to establish a simple, rapid, and accurate method for detecting Vibrio harveyi.

[0004] The main methods for detecting Vibrio harveyi include traditional isolation and culture methods, immunological methods, and molecular biological detection methods, among which molecular biological methods are the most widely used. Although PCR primers and methods exist for detecting Vibrio harveyi, these methods require expensive instruments such as PCR machines, making them unsuitable for rapid on-site testing in grassroots farms. Besides instrument limitations, some primers also suffer from low sensitivity, potentially leading to false negatives in actual testing. Therefore, it is necessary to develop more sensitive and specific products and methods for rapid on-site detection of Vibrio harveyi to monitor the bacteria and prevent vibriosis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing technologies and provide a LAMP primer set and kit for detecting Vibrio harveyi.

[0006] The first objective of this invention is to provide a LAMP primer set for detecting Vibrio harveyi.

[0007] A second objective of this invention is to provide the application of the LAMP primer set in the preparation of products for detecting Vibrio harveyi.

[0008] A third objective of this invention is to provide a kit for detecting Vibrio harveyi.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention designs a set of LAMP primers with high detection sensitivity and specificity for detecting Vibrio harveyi, including a pair of outer primers F3 / B3 and a pair of inner primers FIP / BIP; wherein, the nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.1, the nucleotide sequence of the outer primer B3 is shown in SEQ ID NO.2, the nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.3, and the nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.4.

[0011] As an optional implementation, the 5' end of the inner primer FIP is labeled with biotin.

[0012] Given that the LAMP primer set described in this invention can specifically detect Vibrio harveyi, this invention also seeks to protect the use of the LAMP primer set in the preparation of products for detecting Vibrio harveyi.

[0013] The present invention also provides a kit for detecting Vibrio harveyi, the kit containing the LAMP primer set, namely the outer primer F3 / B3 shown in SEQ ID NO. 1-2 and the inner primer FIP / BIP shown in SEQ ID NO. 3-4.

[0014] Specifically, the kit also contains reagents and colorimetric reagents required for the LAMP reaction.

[0015] More specifically, the colorimetric reagent is SYBR Green I, hydroxynaphthol blue, or calcein-manganese ions.

[0016] More specifically, the reagents required for the LAMP reaction include Mg 2+ , dNTPs and betaine, the Mg 2+ The concentration used is 1.2 mmol·L⁻¹. -1 The concentration of the dNTPs used was 0.64 mmol·L⁻¹. -1 The concentration of betaine used is 0.25 mmol·L⁻¹. -1 .

[0017] The present invention also provides a method for detecting Vibrio harveyi using the above-mentioned kit, comprising the following steps:

[0018] S1. Extract DNA from the sample to be tested;

[0019] S2. Using the DNA obtained in step S1 as a template, the LAMP primers described in this invention, namely the outer primers F3 / B3 shown in SEQ ID NO.1-2 and the inner primers FIP / BIP shown in SEQ ID NO.3-4, are used to perform an amplification reaction;

[0020] S3. Result interpretation.

[0021] Specifically, step S1 can extract DNA from the sample to be tested by boiling in water or using a kit.

[0022] Specifically, the amplification reaction system in step S2 is: 2.5 μL of 10×Bst Buffer, MgSO4 (25 mmol·L⁻¹) -1 )1.2 μL, dNTP (10 mmol·L -1 1.6 μL, inner primer (20 nmol·L⁻¹) -1 1.6 μL of outer primer (10 nmol·L⁻¹) -1 0.2 μL, betaine (5 mmol·L) -1 1.25 μL of Bst DNA polymerase (8 U), 1 μL of DNA template, and 25 μL of DEPC-treated water.

[0023] The above reaction system is the optimal system optimized by this invention, wherein Mg 2+ The optimal concentration for use is 1.2 mmol / L. -1 The optimal concentration of the dNTPs is 0.64 mmol·L⁻¹. -1 The optimal concentration of betaine is 0.25 mmol·L⁻¹. -1 The optimal concentration ratio of the inner and outer primers is 16:1.

[0024] Specifically, the amplification reaction conditions in step S2 are: 60℃~65℃ for 35~60 min.

[0025] Preferably, the reaction conditions are 60°C for 45 min.

[0026] Specifically, when the colorimetric reagent used is SYBR Green I, the result determination steps are as follows: after the reaction is complete, add 1 μL of 1000×SYBR Green I to the reaction tube; if the reaction solution is green, the test result is positive; if the reaction solution is yellow, the test result is negative.

[0027] Specifically, when the colorimetric reagent used is hydroxynaphthol blue (HNB), the result determination procedure is as follows: before the reaction, add 2.08 mmol·L⁻¹ of HNB to the reaction tube. -1HNB; if the reaction solution is blue, the test result is positive; if the reaction solution is blue-purple, the test result is negative.

[0028] Specifically, when the colorimetric reagent used is calcein-manganese ions, the result determination steps are as follows: mix calcein-manganese ions at a concentration ratio of 1.25:1.5, and then add them to the reaction system at an addition ratio of 1:12 (add before the reaction). After the reaction is completed, observe the color of the reaction tube; if the reaction solution is green, the test result is positive; if the reaction solution is yellow, the test result is negative.

[0029] The present invention also provides another kit for detecting Vibrio harveyi, wherein the kit contains, in addition to the LAMP primer set, a transverse flow test strip and reagents required for the transverse flow test strip color development.

[0030] Specifically, the 5' end of the inner primer FIP in the LAMP primer set is biotin-tagged.

[0031] Specifically, the kit also contains a DNA probe, the nucleotide sequence of which is shown in SEQ ID NO.5, and its 5' end is labeled with fluorescein isothiocyanate.

[0032] This invention also provides a method for detecting Vibrio harveyi using the transverse flow test strip, the specific steps of which are as follows:

[0033] S1. Extract DNA from the sample to be tested;

[0034] S2. Using the DNA obtained in step S1 as a template, the LAMP primers described in this invention, namely the outer primers F3 / B3 shown in SEQ ID NO.1-2 and the inner primers FIP / BIP shown in SEQ ID NO.3-4, are used to perform an amplification reaction. The 5' end of the inner primer FIP is labeled with biotin.

[0035] S3. Add FITC-labeled DNA probes to the reaction product obtained from the LAMP reaction. After hybridization at 63°C for 5 min, drop the reaction mixture onto the sample pad of the transverse flow test strip. If both the detection line and the control line turn red, the test result is positive; if the detection line does not turn red but the control line turns red, the test result is negative; if neither the detection line nor the control line changes, the test needs to be repeated.

[0036] The present invention has the following beneficial effects:

[0037] This invention provides a LAMP primer set for detecting Vibrio harveyi. Based on this, the invention also provides a kit and method for detecting Vibrio harveyi. The LAMP primer set described in this invention exhibits high detection specificity and sensitivity, with a limit of detection of 2.4 × 10⁻⁶.0 The copy / μL method for detecting Vibrio harveyi constructed using this method has the advantages of simple operation and accurate detection results. It overcomes the shortcomings of existing molecular biological detection methods for Vibrio harveyi, such as long detection time, low sensitivity, high cost, and difficulty in field application. It is suitable for rapid field detection in grassroots farms and helps in the prevention and control of vibriosis. Attached Figure Description

[0038] Figure 1 The results represent the specificity detection results of the LAMP primer set; where M: Marker; Lane 1: Vibrio harveyi; Lane 2: Vibrio cholerae; Lane 3: Vibrio vulnificus; Lane 4: Vibrio parahaemolyticus; Lane 5: Vibrio alginolyticus; Lane 6: Vibrio fluvialis; Lane 7: Vibrio feniolidone; Lane 8: Lactobacillus plantarum; Lane 9: Streptococcus dolphinus; Lane 10: Streptococcus agalactiae; Lane 11: Staphylococcus aureus; Lane 12: Bacillus licheniformis; Lane 13: Bacillus subtilis; Lane 14: Bacillus cereus; Lane 15: Enterococcus faecalis; Lane 16: Escherichia coli; Lane 17: Proteus mirabilis; Lane 18: Klebsiella pneumoniae; Lane 19: Salmonella enterica; Lane 20: Pseudomonas aeruginosa; Lane 21: Negative control.

[0039] Figure 2 The results represent the optimization of the LAMP reaction system and conditions; where A represents the optimized reaction temperature, B represents the optimized reaction time, and C represents the optimized Mg... 2+ The optimized concentration results are as follows: D represents the optimized concentration of dNTPs, E represents the optimized concentration of betaine, and F represents the optimized concentration ratio of inner and outer primer pairs.

[0040] Figure 3 The results of different detection methods combined with LAMP primer sets are shown below; Sample 1: Vibrio harveyi; Sample 2: Vibrio cholerae; Sample 3: Vibrio vulnificus; Sample 4: Vibrio parahaemolyticus; Sample 5: Vibrio alginolyticus; Sample 6: Vibrio fluvialis; Sample 7: Vibrio feniolidone; Sample 8: Lactobacillus plantarum; Sample 9: Streptococcus dolphinus; Sample 10: Streptococcus agalactiae; Sample 11: Staphylococcus aureus; Sample 12: Bacillus licheniformis; Sample 13: Bacillus subtilis; Sample 14: Bacillus cereus; Sample 15: Enterococcus faecalis; Sample 16: Escherichia coli; Sample 17: Proteus mirabilis; Sample 18: Klebsiella pneumoniae; Sample 19: Salmonella enterica; Sample 20: Pseudomonas aeruginosa; Sample 21: Negative control.

[0041] Figure 4The figures show the sensitivity detection results for PCR primers and LAMP primer sets; the left figure shows the sensitivity detection results for PCR primers, and the right figure shows the sensitivity detection results for LAMP primer sets; lane M: DL 2000 bp; lane 1: 2.4 × 10⁻⁶. 8 copy / μL; Lane 2: 2.4×10 7 copy / μL; Lane 3: 2.4×10 6 copy / μL; Lane 4: 2.4×10 5 copy / μL; Lane 5: 2.4×10 4 copy / μL; Lane 6: 2.4×10 3 copy / μL; Lane 7: 2.4×10 2 copy / μL; Lane 8: 2.4×10 1 copy / μL; Lane 9: 2.4×10 0 copy / μL; Lane 10: 2.4×10 -1 copy / μL; Lane 11: 2.4×10 -2 copy / μL; Lane 12: negative control.

[0042] Figure 5 The results of LAMP primer set detection of grouper samples challenged with different pathogenic bacteria and tilapia samples challenged with Vibrio harveyi are shown below; Lane M: DL 2000 bp; Lane 1: Vibrio harveyi challenged grouper; Lane 2: Vibrio harveyi challenged tilapia; Lane 3: Vibrio alginolyticus challenged grouper; Lane 4: Vibrio fluvibrio challenged grouper; Lane 5: Normal grouper; Lane 6: Normal tilapia; Lane 7: Negative control.

[0043] Figure 6 The results of LAMP primer sets combined with different detection methods were obtained for grouper samples and tilapia samples challenged with different pathogenic bacteria and Vibrio harveyi. Sample 1: grouper challenged with Vibrio harveyi; Sample 2: tilapia challenged with Vibrio harveyi; Sample 3: grouper challenged with Vibrio alginolyticus; Sample 4: grouper challenged with Vibrio fluvii; Sample 5: normal grouper; Sample 6: normal tilapia; Sample 7: negative control. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] Example 1: Design of LAMP Primer Sets

[0047] This invention designed several different sets of LAMP primers for detecting Vibrio harveyi. After specificity and sensitivity testing, the LAMP primer sets shown in Table 1 were ultimately selected to construct the detection kit and method for Vibrio harveyi. To complement the transverse flow test strip, this invention also designed a DNA probe, the sequence of which is shown in Table 1.

[0048] Table 1 Nucleotide sequences of the LAMP primer set

[0049]

[0050] Example 2: Specificity detection of LAMP primer sets

[0051] This embodiment tested the specificity of the LAMP primer sets shown in Table 1, and the information of the strains used is shown in Table 2:

[0052] Table 2. Experimental strains used for specific detection

[0053]

[0054] The specific testing steps are as follows:

[0055] S1. Extract DNA from the sample (bacteria) to be tested;

[0056] DNA can be extracted using the boiling method or a kit; the extraction process of the boiling method is as follows: (1) Take 1-2 mL of bacterial culture medium, centrifuge at 12000 rpm for 1 min, and discard the supernatant; (2) Wash the precipitate twice with 1×PBS, and then suspend the precipitate in 200 μL of sterile water; (3) Boil the bacterial suspension in boiling water for 5-10 min, centrifuge at 12000 rpm for 1 min, and store the supernatant at -20℃.

[0057] The kit can be the bacterial genomic DNA extraction kit from Beijing Tiangen Biotech Co., Ltd. The specific extraction process can be performed according to the kit instructions.

[0058] S2. Using the DNA obtained in step S1 as a template, perform the amplification reaction using the LAMP primers shown in Table 1 (SEQ ID NO. 1-4);

[0059] In this embodiment, DNA extracted from the experimental strain by boiling in water was used as a template for amplification. The amplification reaction system of the LAMP primer set was: 2.5 μL of 10×Bst Buffer and MgSO4 (25 mmol·L⁻¹). -1 )1.2 μL, dNTP (10 mmol·L -11.6 μL, inner primer (20 nmol·L⁻¹) -1 1.6 μL of outer primer (10 nmol·L⁻¹) -1 0.2 μL, betaine (5 mmol·L) -1 1.25 μL of Bst DNA polymerase (8 U), 1 μL of DNA template, and 25 μL of DEPC-treated water.

[0060] The reaction conditions were: 61℃ for 50 min followed by inactivation at 80℃ for 2 min.

[0061] S3. Result determination;

[0062] After the reaction was completed, the reaction products were detected by 2% agarose gel electrophoresis.

[0063] The specificity detection results of the LAMP primer set are as follows: Figure 1 As shown in the figure, M: Marker; Lane 1: Vibrio harveyi; Lane 2: Vibrio cholerae; Lane 3: Vibrio vulnificus; Lane 4: Vibrio parahaemolyticus; Lane 5: Vibrio alginolyticus; Lane 6: Vibrio fluvialis; Lane 7: Vibrio feniolidone; Lane 8: Lactobacillus plantarum; Lane 9: Streptococcus dolphinii; Lane 10: Streptococcus agalactiae; Lane 11: Staphylococcus aureus; Lane 12: Bacillus licheniformis; Lane 13: Bacillus subtilis; Lane 14: Bacillus cereus; Lane 15: Enterococcus faecalis; Lane 16: Escherichia coli; Lane 17: Proteus mirabilis; Lane 18: Klebsiella pneumoniae; Lane 19: Salmonella enterica; Lane 20: Pseudomonas aeruginosa; Lane 21: Negative control. Figure 1 The results show that, except for lane 1 corresponding to Vibrio harveyi, which has a band, the other lanes have no bands, indicating that the LAMP primer set described in this invention has good specificity and can be used for the specific detection of Vibrio harveyi.

[0064] Example 3 Optimization of the reaction system

[0065] The present invention optimizes the reaction system described in Example 2, and the optimization conditions include reaction temperature, reaction time, and Mg. 2+ Concentrations of dNTPs, betaine, and the ratio of inner to outer primer concentrations.

[0066] Optimization of reaction temperature: After preparing the reaction system according to Example 2, it was kept at 60, 61, 62, 63, 64, and 65°C for 50 min, respectively, followed by inactivation at 80°C for 2 min. After the reaction, the results were detected by 2% agarose gel electrophoresis. The optimization results of the LAMP amplification reaction temperature are as follows: Figure 2 As shown in Figure A, the temperatures corresponding to lanes 1 through 6 are 60, 61, 62, 63, 64, and 65℃, respectively. Figure 2 As shown in Figure A, bands can be amplified at all of the above reaction temperatures with no significant difference in effect. Therefore, 60℃ was selected as the optimal reaction temperature.

[0067] Optimization of reaction time: After preparing the reaction system according to Example 2, it was kept at 60℃ for 30, 35, 40, 45, 50, 55, and 60 min respectively, followed by inactivation at 80℃ for 2 min. After the reaction, the results were detected by 2% agarose gel electrophoresis. The optimization results of LAMP amplification reaction time are as follows: Figure 2 As shown in Figure B, the reaction times corresponding to lanes 1-7 are 30, 35, 40, 45, 50, 55, and 60 minutes, respectively. Figure 2 As shown in Figure B, there was no significant difference in the amplified bands obtained at reaction times of 45, 50, 55 and 60 min. Therefore, 45 min was selected as the optimal reaction time.

[0068] Mg 2+ Concentration optimization: Prepare the reaction system according to Example 2, and adjust the Mg content in the system. 2+ Concentrations of 0, 1.2, 2.4, 3.6, 4.8, 6, and 8 mmol / L were used. The mixture was incubated at 60℃ for 45 min, followed by inactivation at 80℃ for 2 min. After the reaction, the results were detected by 2% agarose gel electrophoresis. The Mg in the LAMP reaction system... 2+ The optimization results of the concentration are as follows: Figure 2 As shown in C, the Mg corresponding to lanes 1-7 2+ The concentrations were 0, 1.2, 2.4, 3.6, 4.8, 6, and 8 mmol / L, respectively. Figure 2 As shown in Figure C, Mg 2+ The optimal concentration is 1.2 mmol / L. -1 .

[0069] Optimization of dNTP concentration: The reaction system was prepared according to Example 2, and the dNTP concentrations were adjusted to 0, 0.32, 0.64, 1.28, 1.92, 2.56, and 3.2 mmol / L. The system was incubated at 60°C for 45 min, followed by inactivation at 80°C for 2 min. After the reaction, the results were analyzed by 2% agarose gel electrophoresis. The optimization results of the dNTP concentration in the LAMP reaction system are shown below. Figure 2 As shown in Figure D, the dNTP concentrations corresponding to lanes 1–7 are 0, 0.32, 0.64, 1.28, 1.92, 2.56, and 3.2 mmol / L, respectively. Figure 2 The results shown in Figure D indicate that the optimal concentration of dNTPs is 0.64 mmol / L. -1 .

[0070] Optimization of betaine concentration: The reaction system was prepared according to Example 2, and the betaine concentrations were adjusted to 0, 0.25, 0.5, 1, 1.5, and 2 mmol / L. The system was incubated at 60°C for 45 min, followed by inactivation at 80°C for 2 min. After the reaction, the results were analyzed by 2% agarose gel electrophoresis. The optimization results of the betaine concentration in the LAMP reaction system are as follows: Figure 2 As shown in E, the betaine concentrations corresponding to lanes 1-6 are 0, 0.25, 0.5, 1, 1.5, and 2 mmol / L, respectively. Figure 2 The results shown in E indicate that the optimal concentration of betaine is 0.25 mmol·L⁻¹. -1 .

[0071] Optimization of the primer-inner-primer concentration ratio: The reaction system was prepared according to Example 2, and the primer-inner-primer concentration ratios were adjusted to 1:1, 2:1, 4:1, 8:1, and 16:1. The system was incubated at 60°C for 45 min, followed by inactivation at 80°C for 2 min. After the reaction, the results were detected by 2% agarose gel electrophoresis. The optimized primer-inner-primer concentration ratio in the LAMP reaction system is shown below. Figure 2 As shown in F, the concentration ratios of the inner and outer primers corresponding to lanes 1-5 are 1:1, 2:1, 4:1, 8:1, and 16:1, respectively. Figure 2 As shown in F, the optimal concentration ratio of the inner and outer primers is 16:1.

[0072] Example 4: Kit for detecting Vibrio harveyi

[0073] This invention also provides a kit for detecting Vibrio harveyi, the kit containing the LAMP primer set shown in Table 1, namely, the outer primers F3 / B3 shown in SEQ ID NO. 1-2 and the inner primers FIP / BIP shown in SEQ ID NO. 3-4. The kit also contains reagents required for the LAMP reaction and a colorimetric reagent; the colorimetric reagent is SYBR Green I, hydroxynaphthol blue, or calcein-manganese ions, and the reagents required for the LAMP reaction include Mg... 2+ dNTPs and betaine.

[0074] When using this kit for detection, the reaction system and conditions for the LAMP amplification reaction are the same as in Example 2 or 3.

[0075] When using SYBR Green I as the chromogenic reagent, the activity of Bst DNA polymerase is inhibited because SYBR Green I has a stronger binding affinity to DNA molecules. Therefore, this dye is generally added after the reaction. The method for interpreting results when using SYBR Green I is as follows: After the reaction, add 1 μL of 1000×SYBR Green I to the reaction tube and mix well. If the reaction solution after adding 1000×SYBR Green I turns green, the test result is positive, indicating the presence of Vibrio harveyi in the sample. If the reaction solution turns yellow, the test result is negative.

[0076] When hydroxynaphthol blue (HNB) is used as the colorimetric reagent, its color changes with pH. After the reaction, magnesium ions combine with pyrophosphate ions to form a precipitate, causing a pH change. Therefore, HNB needs to be added to the reaction system before the reaction. The method for determining the results when using HNB is as follows: add HNB to the reaction tube before the reaction to a final concentration of 2.08 mmol / L. -1 HNB; if the reaction solution is blue, the test result is positive; if the reaction solution is blue-purple, the test result is negative.

[0077] When the colorimetric reagent used is calcein-manganese ions, the calcein fluorescent group is quenched before the reaction, resulting in an overall orange color. After the reaction, the manganese ions precipitate, the calcein fluorescent group is released, and the system turns green. This can be used to determine the detection result. The method for determining the result when using calcein-manganese ions is as follows: Mix calcein-manganese ions at a concentration ratio of 1.25:1.5, and then add them to the reaction system at an addition ratio of 1:12 (added before the reaction). After the reaction, observe the color of the reaction tube. If the reaction solution is green, the detection result is positive; if the reaction solution is yellow, the detection result is negative.

[0078] The present invention also provides another kit for detecting Vibrio harveyi, wherein the kit contains, in addition to the LAMP primer set shown in Table 1, a transverse flow test strip and a DNA probe shown in SEQ ID NO. 5; the 5' end of the inner primer FIP in the LAMP primer set is biotin-labeled, and the 5' end of the probe is fluorescein isothiocyanate (FITC)-labeled.

[0079] When using this kit for detection, first perform LAMP amplification using a biotin-labeled LAMP primer set, with the reaction system and conditions the same as in Example 2 or 3. After the reaction, hybridize the probe labeled with fluorescein isothiocyanate with the aforementioned LAMP reaction product at 63°C for 5 min. If the sample contains Vibrio harveyi, its LAMP reaction product will form a complex with the FITC-labeled probe. When the final reaction solution is added to the transverse flow test strip, both the detection line and the control line will turn red. However, if the sample does not contain Vibrio harveyi, the reaction tube will not form a complex. When the final reaction solution is added to the transverse flow test strip, the mixture will pass directly through the detection line, resulting in only the control line turning red. The reaction can be visually determined whether it has occurred.

[0080] The method for determining the results when using the transverse flow test strip is as follows: Add a FITC-labeled DNA probe (nucleotide sequence as shown in SEQ ID NO.5) to the reaction product obtained from the LAMP reaction. After hybridization at 63°C for 5 min, drop the reaction mixture onto the sample pad of the transverse flow test strip. If both the test line and the control line turn red, the test result is positive; if the test line does not turn red but the control line turns red, the test result is negative; if neither the test line nor the control line turns red, the test must be repeated.

[0081] To test the detection effectiveness of the kit described in this invention, this embodiment used the same DNA template as in Example 2, and performed detection using the above kit. The detection results of the LAMP primer set combined with different detection methods are as follows: Figure 3 As shown, by Figure 3 The results show that the LAMP primer set described in this invention can specifically detect Vibrio harveyi, and the detection results are accurate and easy to interpret.

[0082] Example 5 Sensitivity Detection

[0083] DNA was extracted from the liver tissue of a grouper infected with Vibrio harveyi (0.1g). The DNA concentration was measured, and the instrument displayed the DNA unit as ng / µL. This unit was then converted to copies / µL. In this example, the concentration of the extracted DNA template was 2.4 × 10⁻⁶. 8 Copy / μL, and dilute it 10-fold successively to obtain 2.4×10. 8 ~2.4×10 -2 Copy / μL; using this as a template, LAMP and PCR detection were performed respectively, wherein the primers used in the PCR reaction were the external primers F3 / B3 described in this invention.

[0084] The LAMP reaction system is the same as in Example 2 or 3. The PCR reaction system is: 10 µL rTaq, 1 µL each of F3 and B3 primers, 1 µL DNA, and ddH2O to make up to 20 µL.

[0085] The PCR reaction conditions were: 94℃ for 3 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, 72℃ for 5 min, and finally 32 extension cycles.

[0086] After the LAMP and PCR reactions were completed, 5 µL of the reaction products were taken from each reaction and detected by 2% agarose gel electrophoresis to compare the sensitivity of the PCR and LAMP detection methods.

[0087] Sensitivity detection results of PCR primers and LAMP primer sets are as follows: Figure 4 As shown. By Figure 4 The results show that the detection sensitivity of the LAMP primer set described in this invention is 2.4 × 10⁻⁶. 0 copy / μL.

[0088] Example 6: Actual detection of infected fish samples

[0089] Vibrio harveyi cultured overnight (5.8 × 10⁻⁶) 8 Healthy pearl grouper and tilapia were injected with cfu / mL. The healthy pearl grouper were also injected with Vibrio alginolyticus and Vibrio fluvialis cultured overnight at the same concentration. After 24 h of challenge, DNA was extracted from the liver tissue of the fish. The collected samples were tested using LAMP primers, and the detection method was the same as in Example 2.

[0090] The results of LAMP primer set detection of grouper samples challenged with different pathogenic bacteria and tilapia samples challenged with Vibrio harveyi are as follows: Figure 5 As shown; Lane M: DL 2000 bp; Lane 1: Vibrio harveyi challenged grouper; Lane 2: Vibrio harveyi challenged tilapia; Lane 3: Vibrio alginolyticus challenged grouper; Lane 4: Vibrio fluvibrio challenged grouper; Lane 5: Normal grouper; Lane 6: Normal tilapia; Lane 7: Negative control. Figure 5 The results show that the LAMP primer set described in this invention can detect Vibrio harveyi in different fish samples with high specificity and accurate detection results.

[0091] The LAMP primer set, combined with different detection methods, yielded the following results for grouper samples challenged with different pathogenic bacteria and tilapia samples challenged with Vibrio harveyi: Figure 6As shown; among them, Sample 1: Vibrio harveyi challenged grouper; Sample 2: Vibrio harveyi challenged tilapia; Sample 3: Vibrio alginolyticus challenged grouper; Sample 4: Vibrio fluvialis challenged grouper; Sample 5: normal grouper; Sample 6: normal tilapia; Sample 7: negative control. Figure 6 The results show that the LAMP primer set described in this invention, combined with different detection methods, can detect Vibrio harveyi in different fish samples, with high specificity and accurate detection results.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A LAMP primer set for detecting Vibrio harveyi, characterized in that, The primer set includes one pair of outer primers F3 / B3 and one pair of inner primers FIP / BIP. The nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.1, the nucleotide sequence of the outer primer B3 is shown in SEQ ID NO.2, the nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.3, and the nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.

4.

2. The LAMP primer set according to claim 1, characterized in that, The 5' end of the inner primer FIP is labeled with biotin.

3. The use of the LAMP primer set according to claim 1 or 2 in the preparation of products for detecting Vibrio harveyi.

4. A kit for detecting Vibrio harveyi, characterized in that, The kit contains the LAMP primer set as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit also contains reagents and colorimetric reagents required for the LAMP reaction.

6. The reagent kit according to claim 5, characterized in that, The colorimetric reagent is SYBR Green I, hydroxynaphthol blue, or calcein-manganese ions.

7. The reagent kit according to claim 5, characterized in that, The reagents required for the LAMP reaction include Mg 2+ , dNTPs and betaine, the Mg 2+ The concentration used is 1.2 mmol·L⁻¹. -1 The concentration of the dNTPs used was 0.64 mmol·L⁻¹. -1 The concentration of betaine used is 0.25 mmol·L⁻¹. -1 .

8. A kit for detecting Vibrio harveyi, characterized in that, The kit contains the LAMP primer set as described in claim 2.

9. The reagent kit according to claim 8, characterized in that, The kit also contains a transverse flow test strip.

10. The reagent kit according to claim 8, characterized in that, The kit also contains a fluorescein isothiocyanate-labeled DNA probe, the nucleotide sequence of which is shown in SEQ ID NO.5.

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