Primer pair, composition, kit and method for detecting pseudomonas plecoglossicida causing large yellow croaker visceral white-spot disease based on RPA-CRISPR / Cas12a technology

Abstract

The invention relates to a primer pair, a composition, a kit and a method for detecting pseudomonas plecoglossicida causing large yellow croaker visceral white-spot disease based on an RPA-CRISPR / Cas12a technology, the nucleotide sequences of upstream primers RPA-F are respectively as shown in SEQ ID NO.1-4, the nucleotide sequences of downstream primers RPA-R corresponding to the upstream primers RPA-F are respectively as shown in SEQ ID NO.5-8, and the composition comprises the primer pair, crRNA, Cas12a protein and a fluorescent reporter probe. Compared with the prior art, the invention establishes a P. plecoglossicida detection method which is simple and convenient to operate, short in detection time, good in specificity and high in sensitivity and only needs common constant-temperature equipment, and is suitable for on-site rapid detection in basic laboratories and large yellow croaker farms.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a primer pair, composition, kit and method for detecting *Pseudomonas aeruginosa*, which causes visceral white spot disease in large yellow croaker, based on RPA-CRISPR / Cas12a technology. Background Technology

[0002] First discovered in 2000, white spot disease of the viscera of large yellow croaker is caused by *Pseudomonas aeruginosa* (…). Pseudomonas plecoglossicida This disease is caused by *Pseudomonas aeruginosa*. Large yellow croaker infected with *Pseudomonas aeruginosa* exhibit a series of significant clinical symptoms, primarily including white spots on the viscera. These white spots are caused by the accumulation of bacteria and their metabolites, resulting in mold-like white patches on the visceral tissue. In addition, fish infected with *Pseudomonas aeruginosa* show increased mucus secretion on the body surface, gill congestion and hemorrhage, reduced activity, and decreased appetite, severely impacting their growth and survival rate. Studies have shown that *Pseudomonas aeruginosa* is prone to outbreaks under conditions of fluctuating water temperature and excessively high stocking densities. Researchers are working to develop effective diagnostic and control measures to mitigate the impact of this disease on fisheries production.

[0003] Early prevention measures for white spot disease of large yellow croaker include strengthening water quality management, regularly cleaning the aquaculture environment, controlling stocking density, and using antibiotics for preventative treatment. In addition, strengthening quarantine of introduced fish is also a key measure. Currently used diagnostic techniques include PCR, LAMP, and ELISA. Diagnostic methods for *Pseudomonas aeruginosa* mainly include traditional methods (such as blood smears and histopathological analysis), immunological methods (such as enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody detection technology (IFAT),) and molecular biology techniques (including conventional PCR, nested PCR, real-time quantitative PCR, and loop-mediated isothermal amplification (LAMP). These diagnostic techniques each have their advantages and applicable ranges, but they all have high requirements for testing conditions and operators, which is not conducive to large-scale application. Therefore, it is essential to develop a specific, sensitive, rapid, convenient, and easily promoted diagnostic technique.

[0004] RPA-CRISPR / Cas12a is a novel technology that combines recombinase polymerase isothermal amplification with CRISPR / Cas12a technology. Using a pair of upstream and downstream primers typically spaced no more than 500 bp apart, exponential amplification of the target sequence can be achieved within 10-20 minutes in an RPA reaction system. Subsequently, a designed crRNA containing the complementary region of the target fragment guides and activates Cas12a to cleave the ssDNA surrounding the template. At this point, the fluorescent reporter molecule is cleaved, releasing the FAM group. Visible fluorescence can be observed under ultraviolet light after 30 minutes. Combining RPA and CRISPR / Cas12a offers advantages such as ease of operation, low equipment requirements, low cost, and high speed, making it suitable for on-site or production line detection. Summary of the Invention

[0005] The purpose of this invention is to provide a primer pair, composition, kit, and method for detecting *Pseudomonas aeruginosa*, a bacterium that causes visceral white spot disease in large yellow croaker, based on RPA-CRISPR / Cas12a technology. This addresses the problems existing in the prior art. The method of this invention is suitable for rapid detection of visceral white spot disease in large yellow croaker in basic laboratories and large yellow croaker farms, reducing the risk of rapid spread of this newly emerging pathogen, improving the control effect of visceral white spot disease in large yellow croaker, and providing technical support for improving the growth and survival rate of large yellow croaker.

[0006] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of this invention is to provide a primer pair for detecting *Pseudomonas aeruginosa*, which causes visceral white spot disease in large yellow croaker, based on RPA-CRISPR / Cas12a technology. This primer pair is an RPA primer pair for amplifying the *Pseudomonas aeruginosa* DVB73_RS07365 gene, including one of the following a) to d): a) Nucleotide sequences as shown in SEQ ID NO.1 for upstream primer RPA-F1 and as shown in SEQ ID NO.5 for upstream primer RPA-R1; b) Nucleotide sequences as shown in SEQ ID NO.2 for upstream primer RPA-F2 and as shown in SEQ ID NO.6 for upstream primer RPA-R2; c) Nucleotide sequences as shown in SEQ ID NO.3 for upstream primer RPA-F3 and as shown in SEQ ID NO.7 for upstream primer RPA-R3; d) Nucleotide sequences as shown in SEQ ID NO.4 for upstream primer RPA-F4 and as shown in SEQ ID NO.8 for upstream primer RPA-R4.

[0007] The second technical solution of the present invention is to provide a composition for detecting and killing *Pseudomonas aeruginosa* causing visceral white spot disease in large yellow croaker based on RPA-CRISPR / Cas12a technology, comprising primer pairs and crRNA as described in one of the above technical solutions, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO.9.

[0008] In some specific embodiments, the composition further includes a Cas12a protein and a fluorescent reporter probe, wherein the nucleotide sequence of the fluorescent reporter probe is TTATT.

[0009] The third technical solution of the present invention is to provide a kit for detecting Pseudomonas aeruginosa, which causes white spot disease in the viscera of large yellow croaker, based on RPA-CRISPR / Cas12a technology, comprising the primer pair as described in the first technical solution above, and / or comprising the composition as described in the second technical solution above.

[0010] The fourth technical solution of the present invention is to provide the use of the primer pair as described in the first technical solution above, the composition as described in the second technical solution above, and / or the kit as described in the third technical solution above in any of the following: Application in the preparation of products for detecting Pseudomonas aeruginosa, a bacterium that causes visceral white spot disease in large yellow croaker; Application in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by *Pseudomonas aeruginosa* infection; Application in the preparation of products for screening diseases caused by *Pseudomonas aeruginosa* infection.

[0011] The fifth technical solution of the present invention provides a method for detecting and killing *Pseudomonas aeruginosa* causing visceral white spot disease in large yellow croaker based on RPA-CRISPR / Cas12a technology, comprising the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA sample obtained in step S1 as a template, RPA amplification is performed using the primer pair described in one of the above technical solutions to obtain the RPA amplification product. S3. The RPA amplification products obtained in step S2 were detected using a CRISPR / Cas12a cleavage reaction system. S4. Determine the result based on the fluorescence intensity returned under ultraviolet irradiation.

[0012] In some specific implementations, the reaction system for RPA amplification in step S2 is as follows: Add 29.4 μL of buffer A, 2 μL of 10 mM upstream primer, 2 μL of 10 mM downstream primer, 2 μL of DNA template, and 2.5 μL of buffer B to a lyophilized reagent reaction tube. Add dd H2O to bring the volume to 50 μL. The lyophilized reagent reaction tubes, buffer A, and buffer B are derived from a DNA amplification kit. The concentration of the DNA template added is greater than or equal to 10 fg / μL.

[0013] In some specific implementations, the reaction conditions for RPA amplification in step S2 are: incubation at 37°C for (15~20) min.

[0014] In some specific embodiments, step S3, the CRISPR / Cas12a cleavage reaction system is: 2 μL 10xCas12a reaction solution, 2 μL Cas12a protein, 5 μL ddH2O, 4 μL 1 μM crRNA, 2 μL fluorescent reporter probe, wherein the final concentration of Cas12a protein is 100 ng, and the final concentration of fluorescent reporter probe is 2 μM.

[0015] In some specific embodiments, the reaction conditions for step S3, the CRISPR / Cas12a cleavage reaction system, are: incubation at 37°C for (15~60) min.

[0016] Compared with the prior art, the present invention has the following advantages: This invention targets *Pseudomonas aeruginosa* (…). P. plecoglossicida Based on the genome-specific gene DVB73_RS07365, RPA-specific primer pairs and a fluorescent reporter probe were designed. Using RPA-CRISPR / Cas12a technology, a novel detection method was established that is simple to operate, has a short detection time, high specificity, high sensitivity, and requires only ordinary temperature control equipment. P. plecoglossicida The detection methods and products (reagent kits) are suitable for rapid on-site testing in basic laboratories and large yellow croaker farms, and can be effectively used for... P. plecoglossicida Early monitoring and routine testing can reduce the risk of transmission and spread of this newly emerging pathogen, improve the control effect of white spot disease in large yellow croaker viscera, and provide technical support for the detection and diagnosis of white spot disease in large yellow croaker viscera. Attached Figure Description

[0017] Figure 1 This is a schematic flowchart of the detection method of the present invention.

[0018] Figure 2 This is the result of optimizing the RPA primer pairs in the detection method of this invention.

[0019] Figure 3 This is the result of optimizing the CRISPR / Cas12a reaction system in the detection method of this invention.

[0020] Figure 4 This is a specificity analysis diagram of the detection method of the present invention.

[0021] Figure 5 This is a sensitivity analysis diagram of the detection method of the present invention.

[0022] Figure 6 This is a diagram illustrating the on-site detection and analysis of the detection method of the present invention. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0025] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0026] This invention provides a rapid on-site detection method for *Pseudomonas aeruginosa* (…). P. plecoglossicida ) methods, such as Figure 1 As shown.

[0027] The operation process is as follows: Liver, spleen, and kidney tissues were aseptically collected from diseased or dead large yellow croakers suffering from visceral white spot disease. After homogenization, genomic DNA of the pathogen was rapidly extracted using cellulose test strips. The extracted DNA was then amplified by RPA reaction at 37°C for 15 min. Subsequently, a CRISPR / Cas12a cleavage reaction system was added, and the mixture was incubated for 30 minutes to generate a fluorescent signal. The presence or absence of the pathogen could be determined by visually observing green fluorescence under ultraviolet light or by quantitatively detecting the endpoint fluorescence value using a microplate reader.

[0028] Example 1: (1) Based on the gene sequence of Pseudomonas aeruginosa that causes visceral white spot disease in large yellow croaker (DVB73_RS07365) published in GenBank, five upstream primers, five downstream primers, one fluorescent reporter probe, and one crRNA were designed by Primer blast according to the primer design requirements of recombinase polymerase amplification technology. These primer combinations were used to screen for specific amplification of the gene sequence of Pseudomonas aeruginosa that causes visceral white spot disease in large yellow croaker (DVB73_RS07365). All sequences are shown in Table 1. The primers and fluorescent reporter probe were synthesized by Genewiz (Suzhou) Co., Ltd.

[0029] Table 1 shows the primer and probe sequences used. (2) Preparation of positive control samples The *Pseudomonas aeruginosa* was inoculated into 5 mL of TSB medium containing 1‰ Carb resistance and cultured overnight at 30°C. 2 mL of the bacterial culture was taken and the genome of *Pseudomonas aeruginosa* was extracted using a bacterial genome extraction kit (Tiangen DP3021). The DNA concentration was measured using a Nanodrop instrument. The concentration was diluted to 1 ng / μL as a positive control and stored at -20°C for later use.

[0030] (3) The following RPA reaction was performed using a DNA isothermal rapid amplification kit (Weifang Anpu Future Biotechnology Co., Ltd., catalog number WLB8201KIT). 29.4 μL of buffer A, 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, and 2 μL of 1 ng / μL template (final content 2 ng) were added to a lyophilized reagent reaction tube. dd H2O was added to bring the volume to 47.5 μL. Finally, 2.5 μL of buffer B was added to the reaction tube. After thorough mixing, the reaction solution was poured to the bottom of the tube, the cap was slowly closed, and the tube was immediately placed in an isothermal device and incubated at 37°C for 15 min. After the reaction, the reaction tube was removed, and a CRISPR / Cas12a system was added (2 μL of 10 x Cas12a reaction solution, 2 μL of Cas12a protein, 5 μL of ddH2O, 4 μL of 1 μM crRNA, and 2 μL of fluorescent reporter probe, of which 10 x Cas12a was added). The Cas12a reaction solution and Cas12a protein were obtained from Suzhou Xianda Gene Technology Co., Ltd. (product code EM124). The reaction was incubated at 37°C for a period of time. After the reaction was completed, the PCR tubes were placed under a UV lamp to observe the results and the results were recorded by visual observation. Then, the PCR reaction tubes were placed in an ELISA reader to detect the key fluorescence. The wavelength was set to 480 nm and the fluorescence value was recorded.

[0031] (3-1) Regarding RPA upstream and downstream primers: The upstream and downstream primers used in step (4) are the following four groups: RPA-F1 and RPA-R1, RPA-F2 and RPA-R2, RPA-F3 and RPA-R3, RPA-F4 and RPA-R4.

[0032] Other variables remained constant: the CRISPR / Cas12a system consisted of a final content of 100 ng Cas12a, a final concentration of 2 μM fluorescent reporter probe, and incubation at 37°C for 30 minutes.

[0033] The results are as follows Figure 2 As shown, the amplification efficiency of the four sets of RPA upstream and downstream primers was relatively good, among which the RPA-F1 / R1 primer pair returned the highest fluorescence value after the reaction.

[0034] (3-2) Regarding the required final concentrations of Cas12a protein and fluorescent reporter probe in the CRISPR / Cas12a system: The study included two groups: 50 ng Cas12a protein and 1 µM fluorescent reporter probe, and 100 ng Cas12a protein and 2 µM fluorescent reporter probe.

[0035] Other variables remained constant: the upstream and downstream primers for RPA were RPA-F1 and RPA-R1, and the mixture was incubated at 37°C for 30 minutes after being added to the CRISPR / Cas12a system.

[0036] The results are as follows Figure 3 As shown in Figure A, based on visual fluorescence intensity, 100 ng Cas12a protein and 2 µM fluorescent reporter probe were selected as the optimal concentration to ensure clear and distinguishable results.

[0037] Regarding incubation time in the CRISPR / Cas12a system: Optimal reaction time was evaluated at time intervals of 15, 30, 45, and 60 minutes.

[0038] Other variables remained constant: the upstream and downstream primers for RPA were RPA-F1 and RPA-R1, and the CRISPR / Cas12a system included a fluorescent reporter probe with a final content of 100 ng Cas12a and a final concentration of 2 μM.

[0039] The results are as follows Figure 3 As shown in B, incubation at 37°C for 30 minutes produces the maximum signal.

[0040] Therefore, the standard CRISPR / Cas12a reaction system is defined as containing: a final content of 100 ng Cas12a, a final concentration of 2 μM fluorescent reporter probe, and incubation at 37°C for 30 minutes.

[0041] Example 2 Specificity Evaluation DEPC water was set as a negative control, with 1 ng / μL of positive genome ( P. plecoglossicida ) was the positive control, and *Pseudomonas fluorescens* ( P. plecoglossicida ), Pseudomonas aeruginosa ( P. aeruginosa ), Vibrio parahaemolyticus ( V. parahaemolyticus ), Vibrio alginolyticus ( V. alginolyticus ), Vibrio harveyi ( V. harveyi ), Escherichia coli ( E. coli Edwardsiella tarda ( ), E. piscicida Aeromonas salmonidae ( ) A. salmoncida () as a non-target control.

[0042] The following RPA reaction was performed using a DNA isothermal rapid amplification kit (Weifang Anpu Future Biotechnology Co., Ltd., catalog number WLB8201KIT). In a lyophilized reagent reaction tube, 29.4 μL of buffer A, 2 μL of 10 μM RPA-F1, 2 μL of 10 μM RPA-R1, and 2 μL of 1 ng / μL template were added. dd H2O was added to bring the volume to 47.5 μL. Finally, 2.5 μL of buffer B was added to the reaction tube. After thorough mixing, the reaction solution was swished to the bottom of the tube, the cap was slowly closed, and the tube was immediately incubated at 37°C for 15 min. After the reaction, the reaction tube was removed, and the CRISPR / Cas12a system was added (2 μL of 10 x Cas12a reaction solution, 2 μL of Cas12a protein (final concentration 100 ng), 5 μL of ddH2O, 4 μL of 1 μM crRNA, and 2 μL of fluorescent reporter probe (final concentration 2 μM), of which 10 x Cas12a... The Cas12a reaction solution and Cas12a protein were obtained from Suzhou Xianda Gene Technology Co., Ltd. (product code: EM124). The reaction was incubated at 37°C for 30 min. After the reaction, the PCR tubes were placed under a UV lamp to observe the results and the results were recorded by visual observation. Subsequently, the PCR reaction tubes were placed in an ELISA reader to detect the key fluorescence at a wavelength of 480 nm and the fluorescence value was recorded.

[0043] The results are as follows Figure 4 As shown, the negative control (NC) and the DNA of eight pathogens, including *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, *Vibrio parahaemolyticus*, *Vibrio alginolyticus*, *Vibrio harveyi*, *Escherichia coli*, *Edwards ichthyophthirius multifiliis*, and *Aeromonas salmonicidae*, were negative for amplification, while the positive control showed positive genomic DNA detection, indicating that the detection system has good specificity.

[0044] Example 3 Sensitivity Evaluation The genome of *Pseudomonas aeruginosa* was diluted to different initial concentrations (1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL) as a template, and DEPC water was set as a negative control.

[0045] (1) The following RPA reaction was performed using a DNA isothermal rapid amplification kit (Weifang Anpu Future Biotechnology Co., Ltd., catalog number WLB8201KIT). 29.4 μL of buffer A, 2 μL of 10 μM RPA-F1, 2 μL of 10 μM RPA-R1, and 2 μL of template were added to a lyophilized reagent reaction tube. dd H2O was added to bring the volume to 47.5 μL. Finally, 2.5 μL of buffer B was added to the reaction tube. After thorough mixing, the reaction solution was poured to the bottom of the tube, and the tube was slowly capped. The tube was immediately placed in an isothermal device and incubated at 37°C for 15 min. After the reaction, the reaction tube was removed, and a CRISPR / Cas12a system was added (2 μL of 10 x Cas12a reaction solution, 2 μL of Cas12a protein (final concentration 100 ng), 5 μL of ddH2O, 4 μL of 1 μM crRNA, and 2 μL of fluorescent reporter probe (final concentration 2 μM), of which 10 x Cas12a... The Cas12a reaction solution and Cas12a protein were obtained from Suzhou Xianda Gene Technology Co., Ltd. (product code: EM124). The reaction was incubated at 37°C for 30 min. After the reaction, the PCR tubes were placed under a UV lamp to observe the results and the results were recorded by visual observation. Subsequently, the PCR reaction tubes were placed in an ELISA reader to detect the key fluorescence at a wavelength of 480 nm and the fluorescence value was recorded.

[0046] The results are as follows Figure 5 As shown in Figure A, the negative control (NC) and sample 7 (1 fg / μL) showed no fluorescence signal, indicating a negative result. Samples 1–6 (1 ng / μL–10 fg / μL) all showed bands, indicating a positive result. Therefore, the lowest RPA-CRISPR / Cas12a detection limit for this primer-probe combination is determined to be 10 fg / μL.

[0047] (2) In the PCR reaction, the PCR-F1 / R1 primer pair (SEQ ID NO.10, SEQ ID NO.11) in Table 1 was used. The reaction system was as follows: 10 μL of 2x Taq polymerase, 7 μL of ddH2O, 1 μL each of forward and reverse primers, and 1 μL of template. The conditions should be: denaturation temperature 98℃, annealing temperature 60℃, extension temperature 72℃, and cycle number 35.

[0048] After the PCR amplification reaction was completed, 5 μL of the sample from the PCR tube was added to the well of a 1% agarose gel and electrophoresed at 200V for 15 min. Then the bands were observed under ultraviolet light.

[0049] In the PCR sensitivity test, the results are as follows: Figure 5 As shown in B, only (1 ng / μL~10 pg / μL) showed a clear bright band (at 636 bp), while 1 pg / μL-1 fg / μL and the negative control (NC) showed no clear band. Therefore, the lowest PCR sensitivity of this primer-probe combination can be determined to be 10 pg / μL (16.5 copies / μL).

[0050] Therefore, the sensitivity detection line of the RPA-CRISPR / Cas12a primer pair of this invention is 1000 times higher than that of PCR, and also superior to the sensitivity reported in the existing patent CN119753176A (2.34 × 10⁻⁶). 2 The results (copies / μL) demonstrate that this method has good sensitivity.

[0051] Example 4: Clinical Sample Testing To evaluate the clinical applicability of the RPA-CRISPR / Cas12a detection system of this invention, it was tested at 16-20°C with 10 μL of Pseudomonas aeruginosa. 3 CFU ml -1 Large yellow croaker were infected, and death was observed 4 to 9 days after infection.

[0052] From healthy fish ( Figure 6 A) or dead fish ( Figure 6 Tissue samples (spleen, liver, and kidney) were collected in B), homogenized in 600 µL PBS, and rapid genomic DNA was extracted using cellulose test strips. The extracted DNA was directly used for RPA-CRISPR / Cas12a reactions, and the fluorescence output served as a diagnostic reading for infection.

[0053] The following RPA reaction was performed using a DNA isothermal rapid amplification kit (Weifang Anpu Future Biotechnology Co., Ltd., catalog number WLB8201KIT). In a lyophilized reagent reaction tube, 29.4 μL of buffer A, 2 μL of 10 μM RPA-F1, 2 μL of 10 μM RPA-R1, and 2 μL of template were added. dd H2O was added to bring the volume to 47.5 μL. Finally, 2.5 μL of buffer B was added, and the mixture was thoroughly mixed. The reaction solution was then poured to the bottom of the tube, and the tube was slowly capped. The tube was immediately incubated at 37°C for 15 min. After the reaction, the tube was removed, and a CRISPR / Cas12a system was added (2 μL of 10 x Cas12a reaction solution, 2 μL of Cas12a protein (final concentration 100 ng), 5 μL of ddH2O, 4 μL of 1 μM crRNA, and 2 μL of fluorescent reporter probe (final concentration 2 μM), of which 10 x Cas12a... The Cas12a reaction solution and Cas12a protein were obtained from Suzhou Xianda Gene Technology Co., Ltd. (product code: EM124). The reaction was incubated at 37°C for 30 min. After the reaction, the PCR tubes were placed under a UV lamp to observe the results and the results were recorded by visual observation. Subsequently, the PCR reaction tubes were placed in an ELISA reader to detect the key fluorescence at a wavelength of 480 nm and the fluorescence value was recorded.

[0054] according to Figure 6 The results showed that the RPA-CRISPR / Cas12a detection method of the present invention could successfully detect the presence of the pathogen *Pseudomonas aeruginosa* in the spleen, liver and kidney of diseased and dead fish, proving the practicality of the present invention.

[0055] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A primer pair based on RPA-CRISPR / Cas12a technology for detecting and killing *Pseudomonas aeruginosa*, which causes visceral white spot disease in large yellow croaker, characterized in that... For amplifying the *Pseudomonas aeruginosa* DVB73_RS07365 gene, the RPA primer pair includes one of the following a) to d): a) Nucleotide sequences as shown in SEQ ID NO.1 for upstream primer RPA-F1 and as shown in SEQ ID NO.5 for upstream primer RPA-R1; b) Nucleotide sequences as shown in SEQ ID NO.2 for upstream primer RPA-F2 and as shown in SEQ ID NO.6 for upstream primer RPA-R2; c) Nucleotide sequences as shown in SEQ ID NO.3 for upstream primer RPA-F3 and as shown in SEQ ID NO.7 for upstream primer RPA-R3; d) Nucleotide sequences as shown in SEQ ID NO.4 for upstream primer RPA-F4 and as shown in SEQ ID NO.8 for upstream primer RPA-R4.

2. A composition for detecting and killing *Pseudomonas aeruginosa* causing visceral white spot disease in large yellow croaker based on RPA-CRISPR / Cas12a technology, characterized in that, Includes the primer pair as described in claim 1 and crRNA, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO.

9.

3. The composition according to claim 2, characterized in that, The composition also includes the Cas12a protein and a fluorescent reporter probe, wherein the nucleotide sequence of the fluorescent reporter probe is TTATT.

4. A kit for detecting *Pseudomonas aeruginosa* causing visceral white spot disease in large yellow croaker based on RPA-CRISPR / Cas12a technology, comprising the primer pair as described in claim 1, and / or the composition as described in claim 2.

5. The use of the primer pair as described in claim 1, the composition as described in any one of claims 2 or 3, and / or the kit as described in claim 4 in any of the following: Application in the preparation of products for detecting Pseudomonas aeruginosa, a bacterium that causes visceral white spot disease in large yellow croaker; Application in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by *Pseudomonas aeruginosa* infection; Application in the preparation of products for screening diseases caused by *Pseudomonas aeruginosa* infection.

6. A method for detecting and killing *Pseudomonas aeruginosa* causing visceral white spot disease in large yellow croaker based on RPA-CRISPR / Cas12a technology, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA sample obtained in step S1 as a template, RPA amplification is performed using the primer pair as described in claim 1 to obtain the RPA amplification product. S3. The RPA amplification products obtained in step S2 were detected using a CRISPR / Cas12a cleavage reaction system. S4. Determine the result based on the fluorescence intensity returned under ultraviolet irradiation.

7. The method according to claim 6, characterized in that, In step S2, the reaction system for RPA amplification is as follows: Add 29.4 μL of buffer A, 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 2 μL of DNA template, and 2.5 μL of buffer B to a lyophilized reagent reaction tube. Add dd H2O to bring the volume to 50 μL. Among them, the lyophilized powder reagent reaction tubes, buffer A, and buffer B are derived from the DNA amplification kit; The concentration of the DNA template added is greater than or equal to 10 fg / μL.

8. The method according to claim 6, characterized in that, In step S2, the reaction conditions for RPA amplification are: incubation at 37°C for 15-20 min.

9. The method according to claim 6, characterized in that, In step S3, the CRISPR / Cas12a cleavage reaction system consisted of: 2 μL 10x Cas12a reaction solution, 2 μL Cas12a protein, 5 μL ddH2O, 4 μL 1 μM crRNA, and 2 μL fluorescent reporter probe. The final concentration of Cas12a protein was 100 ng, and the final concentration of the fluorescent reporter probe was 2 μM.

10. The method according to claim 6, characterized in that, In step S3, the reaction conditions for the CRISPR / Cas12a cleavage reaction system are: incubation at 37°C for (15~60) min.

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