Primer and kit for rapidly detecting aeromonas salmonicida through RPA-CRISPRCas12a and application

Abstract

The invention discloses a primer and a kit for rapidly detecting aeromonas salmonicida through RPA-CRISPRCas12a and application of the primer and the kit, and relates to the technical field of aquaculture disease prevention and control. The invention solves the problems that the existing detection method is time-consuming and labor-consuming, is easy to cause pollution and needs to be matched with professional detection equipment. The RPA amplification primer disclosed by the invention consists of a forward primer fstA-RPA-F2 and a reverse primer fstA-RPA-R2. The kit disclosed by the invention contains the RPA amplification primer and the crRNA. The method for rapidly detecting the aeromonas salmonicida comprises a test paper method and a fluorescence method. The method disclosed by the invention has the advantages of good inclusiveness, strong specificity, high sensitivity and the like, detection is carried out based on the RPA primer and the crRNA, and the obtained RPA product is detected by using a lateral flow chromatography test strip and fluorescence, so that the purpose of portable visual rapid detection on site or in a culture base is achieved, and the method has a wide application prospect. The method has very high clinical application value in the aspects of early discovery of diseases, epidemiological investigation and comprehensive prevention and control.

Description

Technical Field

[0001] This invention relates to the field of aquaculture disease prevention and control technology, and in particular to primers, kits and applications for rapid detection of Aeromonas salmonicidae using RPA-CRISPRCas12a. Background Technology

[0002] Aeromonas salmonicida, belonging to the genus Aeromonas, is one of the main pathogens causing furunculosis in cold-water fish. This bacterium not only infects salmonids and trout but also various warm-water fish, leading to typical furunculosis symptoms such as furuncles on the body surface, intestinal swelling, and inflammation. This results in high mortality rates and severe economic losses to the aquaculture industry.

[0003] Currently, bacterial pathogen detection is the most common method for detecting Aeromonas salmonidae. However, this method is time-consuming, labor-intensive, prone to contamination, and requires specialized equipment, which seriously hinders the healthy development of aquaculture. Summary of the Invention

[0004] To address the problems of existing detection methods being time-consuming, labor-intensive, prone to contamination, and requiring specialized testing equipment, this invention provides primers, a kit, and applications for rapid detection of Aeromonas salmonicidae using RPA-CRISPRCas12a.

[0005] The RPA amplification primers for rapid detection of Aeromonas salmonicidae using RPA-CRISPRCas12a in this invention consist of a forward primer fstA-RPA-F2 and a reverse primer fstA-RPA-R2, wherein the forward primer fstA-RPA-F2 is 5'-CCAGGAGCCGAGCCAGTAGGCATGATTGGTGA-3', and the reverse primer is 5'-CAAAAGTGATGGCCAAGCTGGGGCTGGAATACG-3'.

[0006] This invention provides a rapid RPA-CRISPRCas12a detection method for the crRNA sequence fstA-crRNA2 of Aeromonas salmonicida, which is 5'-UAAUUUCUACUAAGUGUAGAUUCGGCAAGCGCUAUGUCACCG-3'.

[0007] The RPA-CRISPRCas12a rapid detection kit of Aeromonas salmonicidae of the present invention contains the above-mentioned RPA amplification primers and crRNA.

[0008] The specific steps of the method for rapid detection of Aeromonas salmonicidae of the present invention are as follows:

[0009] I. Prepare a total RPA reaction system of 50 μL: 29.5 µL of RPA reaction reagent Primer Free Rehydration Buffer, 2.4 µL of primer fstA-RPA-F2 with a concentration of 10 µmol / L, 2.4 µL of primer fstA-RPA-R2 with a concentration of 10 µmol / L, 2.5 µL of magnesium acetate with a concentration of 280 mmol / L (mM), 1 µL of DNA template of the sample to be tested, and the remainder Nuclease-free H2O;

[0010] II. RPA isothermal amplification: The reaction conditions were 39℃ for 20 min to obtain RPA amplification products;

[0011] III. RPA-CRISPR / Cas12a reaction: Prepare the CRISPR reaction system and react at 37℃ for 30 min; the CRISPR reaction system consists of: 2 μL Cas12a Reaction buffer 10x, 1 μL probe at a concentration of 5 μmol / L, 1 μL Cas12a protein at a concentration of 1 μmol / L (μM), 1 μL of the above-mentioned crRNA (fstA-crRNA2) at a concentration of 1 μmol / L, 4 μL RPA amplification product, and 11 μL Nuclease-free H2O;

[0012] IV. Determine the results by observing the fluorescence curve or the color change of the product in the tube;

[0013] The probe sequence and fluorescent group are as follows: the fluorescent ssDNA reporter sequence is 5'-FAM-TTTAATTT-BHQ1-3'.

[0014] Further, the preparation method of the total RPA reaction system in step one is as follows: Add all components except magnesium acetate and DNA template to the reaction tube containing RPA reaction reagent powder, vortex thoroughly to mix, centrifuge, open the reaction tube, add 2.5 μL of magnesium acetate to the cap of the reaction tube, then add template DNA to the reaction tube, mix thoroughly and centrifuge.

[0015] The specific steps of the method for rapid detection of Aeromonas salmonicidae of the present invention are as follows:

[0016] I. Prepare a total RPA reaction system of 50 μL: 29.5 µL of RPA reaction reagent Primer Free Rehydration Buffer, 2.4 µL of primer fstA-RPA-F2 with a concentration of 10 µmol / L, 2.4 µL of primer fstA-RPA-R2 with a concentration of 10 µmol / L, 2.5 µL of magnesium acetate with a concentration of 280 mmol / L, 1 µL of DNA template of the sample to be tested, and the remainder Nuclease-free H2O;

[0017] II. RPA isothermal amplification: The reaction conditions were 39℃ for 20 min to obtain RPA amplification products;

[0018] III. CRISPR / Cas12a reaction: Prepare the CRISPR reaction system and react at 37℃ for 30 min; the CRISPR reaction system consists of: 2 μL Cas12a Reaction buffer 10x, 1 μL probe at a concentration of 1 μmol / L, 1 μL Cas12a protein at a concentration of 1 μmol / L, 1 μL of the above-mentioned crRNA (fstA-crRNA2) at a concentration of 1 μmol / L, 4 μL RPA amplification product, and 11 μL Nuclease-free H2O;

[0019] 4. Take 10 μL of CRISPR / Cas12a reaction product and add it to a 1.5 mL centrifuge tube containing 40 μL ddH2O. After mixing, put the test strip in and wait 5~10 min to get the test strip detection result.

[0020] The probe sequence and fluorescent group are as follows: The sequence of the test strip reporter is 5'-FAM-TTTAATTT-Biotin-3'.

[0021] Further, the preparation method of the total RPA reaction system in step one is as follows: Add all components except magnesium acetate and DNA template to the reaction tube containing RPA reaction reagent powder, vortex thoroughly to mix, centrifuge, open the reaction tube, add 2.5 μL of magnesium acetate to the cap of the reaction tube, then add template DNA to the reaction tube, mix thoroughly and centrifuge.

[0022] The beneficial effects of this invention are:

[0023] 1. High sensitivity: Using serially diluted fstA-pMD™19 plasmid as a template, the results showed that the limit of detection (LOD) of this method for the test strip was 10. 2 Copies / test, with fluorescent types as low as 1 copy / test.

[0024] 2. High Specificity: Cross-reactivity tests were performed on nine common pathogens in freshwater fish farming (including Aeromonas salmonidae, Aeromonas tempera, Aeromonas velutipes, Aeromonas hydrophila, Yersinia rumenii, Escherichia coli, Aeromonas salmonidae, Vibrio parahaemolyticus, and Vibrio alginolyticus), and all results were negative. Only Aeromonas salmonidae showed a positive result, fully demonstrating that the crRNA (fstA-crRNA2) and detection system described in this invention have high specificity and no cross-reactivity.

[0025] 3. Reliable verification using actual samples: Under simulated real-world infection scenarios, Aeromonas salmonicidae was added to healthy rainbow trout tissue samples (liver / spleen / kidney / muscle), resulting in a bacterial count of 4.2 × 10⁻⁶. 6 Simulated clinical samples at CFU / mg were used. The method was applied for detection, and the results showed that all 10 simulated infection samples were accurately detected as positive, while the blank control was negative, achieving a detection rate of 100%. This demonstrates the applicability of the method to complex real-world samples.

[0026] 4. Technical Advantages and Application Value: Comprehensive verification results demonstrate that the RPA-CRISPR / Cas12a rapid detection technology of this invention possesses advantages such as high accuracy, high sensitivity, and strong specificity. It combines the rapid isothermal amplification of RPA with the high specificity recognition and signal amplification capabilities of CRISPR / Cas12a, achieving efficient, specific, and accurate detection of target pathogens. This invention is compatible with both fluorescence reading and lateral flow strip (LFS) result interpretation modes, enabling rapid and visual detection without relying on expensive specialized instruments and complex laboratory conditions. This provides a rapid diagnostic tool for the early detection, epidemiological investigation, and comprehensive prevention and control of furuncles caused by Aeromonas salmoneri, and has high clinical application value.

[0027] The method described in this invention has the advantages of good inclusiveness, high specificity, and high sensitivity. Based on the RPA primers and crRNA, the obtained RPA product is detected using lateral flow chromatography test strips and fluorescence detection, realizing the purpose of portable, visual, and rapid detection in the field or breeding base. It has high clinical application value in the early detection of diseases, epidemiological investigation, and comprehensive prevention and control. Attached Figure Description

[0028] Figure 1 Electrophoresis of the isothermal amplification and purification products was performed using primers for the fstA gene.

[0029] Figure 2 CRISPR / Cas12a detection of the fstA gene;

[0030] Figure 3 Fluorescence amplification curves for screening fstA gene crRNA;

[0031] Figure 4 Electrophoresis was used for PCR identification of the fstA gene in bacterial culture.

[0032] Figure 5 Electrophoresis for RPA amplification time selection and temperature selection for the fstA gene;

[0033] Figure 6 CRISPR / Cas12a detection (Reporter dosage);

[0034] Figure 7 Optimize the concentrations of crRNA (fstA-crRNA2) and Cas12a protein;

[0035] Figure 8 CRISPR / Cas12a detection of the fstA gene (fluorescence method and test strip method);

[0036] Figure 9 CRISPR / Cas12a detection of the fstA gene (fluorescence method and test strip method);

[0037] Figure 10 CRISPR / Cas12a detection of the fstA gene (fluorescence method and test strip method). Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0040] Example 1: The primers described in this invention were used for rapid detection of Aeromonas salmonicidae using RPA-CRISPR / Cas12a. The experimental method is as follows:

[0041] I. Prepare a total RPA reaction system of 50 μL: 29.5 µL of RPA reaction reagent Primer-Free Rehydration Buffer, 2.4 µL of upstream primer with a concentration of 10 µmol / L, 2.4 µL of downstream primer with a concentration of 10 µmol / L, 2.5 µL of magnesium acetate with a concentration of 280 mmol / L, 1 µL of DNA template of the sample to be tested, and the remainder Nuclease-free H2O;

[0042] II. RPA isothermal amplification: The reaction conditions were 39℃ for 20 min to obtain RPA amplification products;

[0043] III. CRISPR / Cas12a Reaction: Configure the CRISPR reaction system and react at 37℃ for 30 min; the CRISPR reaction system consists of: 2 μL Cas12a Reaction buffer 10x, 1 μL probe with a concentration of 5 μmol / L, 1 μL Cas12a protein with a concentration of 1 μmol / L, 1 μL crRNA (fstA-crRNA2) with a concentration of 1 μmol / L, 4 μL RPA amplification product, and 11 μL Nuclease-free H2O;

[0044] IV. Determine the results by observing the fluorescence curve or the color change of the product in the tube;

[0045] The probe sequences and fluorescent groups are as follows: the fluorescent ssDNA reporter sequence is 5'-FAM-TTTAATTT-BHQ1-3', and the test strip reporter sequence is 5'-FAM-TTTAATTT-Biotin-3'.

[0046] The rapid detection of the crRNA sequence (fstA-crRNA2) of Aeromonas salmonicidae using RPA-CRISPRCas12a is 5'-UAAUUUCUACUAAGUGUAGAUUCGGCAAGCGCUAUGUCACCG-3'.

[0047] This embodiment selects the conserved region sequence of the *Aeromonas salmonicida* fstA gene. Using the sequence X87995.1 from the NCBI database as a template, and based on the RPA primer design principle, specific primer pairs with amplification fragments of 100-250 bp within the conserved region of the gene were designed online. Following the design principle of PAM recognition and targeting DNA binding to activate Cas12a, crRNA was designed in the region near the PAM site in the targeted amplification fragment. Simultaneously, based on the RPA amplification primer positions, a PCR amplification primer fragment of approximately 2121 bp containing the RPA amplification product was designed to construct the fstA-pMD™19 plasmid.

[0048] Five pairs of RPA amplification primers were designed based on the selected target gene (fstA) in its conserved region (including the PAM region) (as shown in Table 2), and were verified and screened using Aeromonas salmonidae DNA as a template.

[0049] Figure 1Electrophoresis was performed to test the isothermal amplification and purification products of the fstA gene primers. M: Marker DL2000; lanes F1 / R1-F5 / R5 contain the five pairs of primers for the fstA gene, namely fstA-RPA-F1 / R1, fstA-RPA-F2 / R2, fstA-RPA-F3 / R3, fstA-RPA-F4 / R4, and fstA-RPA-F5 / R5; lane P is the positive control; and lane N is the negative control.

[0050] Depend on Figure 1 The results showed that the positive control was positive and the negative control showed no band, indicating that the amplification results were accurate and reliable. All five primer pairs for the fstA gene amplified bright, single target bands of the correct size, making them usable. Among them, primer pair fstA-RPA-F2 / R2 amplified the brightest band and showed the best performance.

[0051] Table 1. Information on RPA and PCR primers

[0052]

[0053] Example 2. The difference between this example and Example 1 is that in step one, the upstream primer is fstA-RPA-F2 and the downstream primer is gldD-RPA-R2; in step two, the corresponding crRNA sequences and primers are designed in the PAM region of the upstream and downstream primers for RPA amplification of the fstA gene. The crRNA sequences are shown in Table 2, namely fstA-crRNA1, fstA-crRNA2 (in this invention), fstA-crRNA3, fstA-crRNA4, and fstA-crRNA5.

[0054] Table 2 crRNA sequences

[0055]

[0056] Corresponding crRNA sequences containing PAM regions were designed and synthesized in the upstream and downstream primer regions of the fstA gene RPA amplification. The RPA amplification products were used as templates for verification and screening using a CRISPR / Cas12a reaction system. Figure 2 For: CRISPR / Cas12a detection of the fstA gene, Figure 3 Fluorescence amplification curves for screening crRNA of the fstA gene. (Source: [Insert source here]) Figure 2 and 3 As can be seen, all five reactions can produce fluorescent signals, but fstA-crRNA2 has the strongest signal. Therefore, fstA-crRNA2 was selected for subsequent experimental testing.

[0057] Example 3 Performance Verification Test

[0058] 1. Plasmid construction

[0059] In this embodiment, PCR amplification primers fstA-PCR-F1 and fstA-PCR-R1 containing RPA amplification products were designed at both ends of the screened RPA amplification primers fstA-RPA-F2 / R2 (the expected amplification fragment size is 2121 bp), and the fstA-pMD™19 plasmid was constructed for subsequent performance verification tests.

[0060] Figure 4 Electrophoresis for PCR identification of the fstA gene in bacterial culture. In the figure, M: Marker DL2000, lanes 1-12 show the PCR identification results of the fstA gene in bacterial culture; lanes 1-2 show the gel recovery electrophoresis results of the fstA gene. Figure 4 As can be seen, a single bright target band of about 2000 bp was obtained, which is consistent with the size of the target fragment predicted by the primers. Sequencing comparison of the amplified product confirmed that it was the fstA gene fragment of Aeromonas salmonidae, indicating that the plasmid was successfully constructed.

[0061] 2. RPA amplification system

[0062] The fstA gene can be amplified via RPA at both 16-24 min and 35-43℃. Therefore, based on a comprehensive comparison of electrophoresis band intensity and fluorescence signal acquisition time, the amplification times were set to 16 min, 18 min, 20 min, 22 min, and 24 min, and the temperatures to 35℃, 37℃, 39℃, 41℃, and 43℃. Using Aeromonas salmonida DNA as a template, the optimal combination of amplification time and temperature was screened according to the RPA amplification system of the kit. Figure 5 Electrophoresis for RPA amplification time selection and temperature selection of the fstA gene were performed. Figure 5 It is evident that selecting 39 ℃ and amplifying for 20 minutes yields the best results.

[0063] Depend on Figure 5 It can be seen that the fstA gene can be amplified by RPA at 16-24 min and 35-43℃. Therefore, based on the comprehensive comparison of electrophoresis band intensity and fluorescence signal acquisition time, 39℃ and 20 min of amplification were selected for subsequent testing.

[0064] 3. Probe, crRNA (fstA-crRNA2) concentration, and Cas12a protein.

[0065] 3.1 Optimization of Cas12a protein and crRNA (fstA-crRNA2) concentrations in the test strip detection system: The concentrations of Cas12a protein and crRNA were adjusted in equal proportions (1:1) to 0.5, 1.0, 1.5, and 2.0 μM (μmol / L), respectively. The concentrations at 10 μM were then analyzed using the RPA-CRISPR / Cas12a system. 4 The reaction was performed using copies / μL of Aeromonas salmonicidae positive plasmid, and the mixture was incubated at 37 °C for 30 min. The results of the reaction with different concentrations of Cas12a protein and crRNA on the test strip were recorded.

[0066] 3.2 The reporter probe is a key factor affecting the detection of CRISPR / Cas12a test strips. Optimization of the reporter probe concentration in the test strip detection system: The concentrations of 0.25, 0.5, 1, 2, and 3 μM (μmol / L) test strip probes were compared with those of 10 μM (μmol / L) using the RPA-CRISPR / Cas12a-LFS system. 4 Different test results were obtained after reacting with copies / μL of Aeromonas salmonicidae positive plasmid.

[0067] Using RPA amplification products (plasmids) as templates, concentration gradients were set to screen the amount of Reporter, and the optimal concentration was selected based on the positive intensity of the test line on the test strip.

[0068] Figure 6 For CRISPR / Cas12a detection (Reporter dosage). Figure 7 To optimize the concentrations of crRNA and Cas12a protein, from Figure 6 It is evident that, based on the test strip results, a darker test line indicates a stronger positive result, and the optimal Reporter dosage is 2 μM. Figure 7 As can be seen from the fluorescence results, the fluorescence is brightest when the concentration of both crRNA and Cas12a protein is 1 μM. Therefore, the optimal concentration of crRNA and Cas12a is 1 μM.

[0069] 4. Sensitivity

[0070] The fstA-pMD™19 plasmid was serially diluted to 10⁻⁶. 5 copies, 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 10 0Using the fstA-pMD™19 plasmid copies as a template, RPA amplification was performed using RPA amplification reagents. After purification of the RPA amplification product, CRISPR / Cas12a detection was performed to test the sensitivity of this method. Figure 8 CRISPR / Cas12a detection of the fstA gene (fluorescence method and test strip method) yielded the following results: Figure 8 As shown, the fluorescence detection sensitivity is 10. 0 copies / test; the sensitivity of the test strip method is 10. 2 copies / test.

[0071] 5. Specificity

[0072] Nine common freshwater fish pathogens, including *Aeromonas salmonicida*, *Aeromonas sobria*, *Aeromonas veronii*, *Aeromonas hydrophila*, *Yersinia ruckeri*, *Escherichia coli*, *Flavobacterium psychrophilum*, *Vibrio parahaemolyticus*, and *V. alginolyticus*, were selected for RPA amplification and then subjected to further testing.

[0073] CRISPR / Cas12a fluorescence detection, nucleic acid detection, and test strip detection were used to verify the specificity of crRNA (fstA-crRNA2) for CRISPR / Cas12a detection of Aeromonas salmonicida. Pathogen information is shown in Table 3.

[0074] Table 3 Information on pathogens

[0075]

[0076] Genomic DNA was extracted from common freshwater fish pathogens such as Aeromonas salmonidus, Aeromonas tempera, Aeromonas vernix, Aeromonas hydrophila, Yersinia rumeni, Escherichia coli, Flavobacterium psychrophilum, Vibrio parahaemolyticus, and Vibrio alginolyticus as templates for specific detection. Figure 9 For the detection of the fstA gene by CRISPR / Cas12a (fluorescence method and test strip method), by Figure 9 As can be seen, Aeromonas salmonidae tested positive, while other strains were negative, and the specificity test results were in line with expectations.

[0077] 6. Actual sample testing

[0078] Using healthy rainbow trout tissue as samples, Aeromonas salmonidae and sterile PBS were added. After tissue homogenization, the supernatant was collected by centrifugation. DNA was extracted from the tissue samples and subjected to RPA amplification, CRISPR / Cas12a detection, nucleic acid testing, and test strip detection. After the fluorescence detection reaction was completed, visual observation was performed under the illumination of a blue LED in a gel imaging system. After the test strip reaction was completed, the test strip results were observed and recorded.

[0079] The *Aeromonas salmonii* strain was revived, and its concentration after shaking was measured to be 4.2 × 10⁻⁶. 9 CFU / mL, 50 mg of healthy rainbow trout tissue samples (liver / spleen / kidney / muscle) were selected. 100 μL of the Aeromonas salmonicidal solution was diluted 10-fold and added to the 50 mg tissue sample to prepare a tissue sample with a bacterial count of 4.2 × 10⁻⁶ CFU / mL. 6 CFU / mg was used to extract tissue DNA, and 10 clinical samples simulating actual infection with Aeromonas salmonida were tested. Figure 10 For the detection of the fstA gene by CRISPR / Cas12a (fluorescence method and test strip method), by Figure 10 As can be seen, the blank control was negative, and all the samples to be tested were positive, with a detection rate of 100%, which is in line with expectations.

[0080] The primers provided in this invention enable a rapid detection method for Aeromonas salmonicida using RPA-CRISPR / Cas12a technology. The core performance parameters and advantages of this method are summarized below:

[0081] 1. Detection Procedure and Conditions: The total detection time for this method is approximately 60 minutes. The RPA isothermal amplification step is performed at 39℃, with an optimized reaction time of 20 minutes. The CRISPR / Cas12a detection step is incubated at a constant temperature of 37℃ for 30 minutes. The optimal amounts of key reagents were determined as follows: Reporter probe concentration 2 μM, crRNA and Cas12a protein concentrations both 1 μM, and the crRNA with the strongest signal (fstA-crRNA2) was selected for specific recognition.

[0082] 2. High sensitivity: Using serially diluted fstA-pMD™19 plasmid as a template, the results showed that the limit of detection (LOD) of this method for the test strip was 10. 2 Copies / test, with fluorescent types as low as 1 copy / test.

[0083] 3. High Specificity: Cross-reactivity tests were performed on nine common pathogens in freshwater fish farming (including Aeromonas salmonidae, Aeromonas tempera, Aeromonas velutipes, Aeromonas hydrophila, Yersinia rumenii, Escherichia coli, Flavobacterium psychrophilum, Vibrio parahaemolyticus, and Vibrio alginolyticus), and all results were negative. Only Aeromonas salmonidae showed a positive result, fully demonstrating that the designed crRNA (fstA-crRNA2) and the detection system have high specificity and no cross-reactivity.

[0084] 4. Reliable Verification Based on Actual Samples: Under simulated actual infection scenarios, Aeromonas salmonicidae was added to healthy rainbow trout tissue samples (liver / spleen / kidney / muscle), resulting in a bacterial count of 4.2 × 10⁻⁶. 6 Simulated clinical samples at CFU / mg were used. The method was applied, and all 10 simulated infection samples were accurately detected as positive, while the blank control was negative, achieving a detection rate of 100%. This demonstrates the applicability of this method to complex real-world samples.

[0085] 5. Technical Advantages and Application Value: Comprehensive validation results demonstrate that this RPA-CRISPR / Cas12a rapid detection technology possesses advantages such as high accuracy, high sensitivity, and strong specificity. It combines the rapid isothermal amplification of RPA with the high specificity recognition and signal amplification capabilities of CRISPR / Cas12a, achieving efficient, specific, and accurate detection of target pathogens. This method is compatible with both fluorescence reading and lateral flow strip (LFS) result interpretation modes, enabling rapid and visual detection without relying on expensive specialized instruments and complex laboratory conditions. This provides a rapid diagnostic tool for the early detection, epidemiological investigation, and comprehensive prevention and control of furuncles caused by Aeromonas salmoneri, possessing high clinical application value.

Claims

1. RPA primers for rapid detection of Aeromonas salmonicidae using RPA-CRISPRCas12a, characterized in that, The RPA amplification primers for rapid detection of Aeromonas salmonicidae using RPA-CRISPRCas12a consist of a forward primer fstA-RPA-F2 and a reverse primer fstA-RPA-R2. The forward primer fstA-RPA-F2 is 5'-CCAGGAGCCGAGCCAGTAGGCATGATTGGTGA-3', and the reverse primer is 5'-CAAAAGTGATGGCCAAGCTGGGGCTGGAATACG-3'.

2. A crRNA for rapid detection of Aeromonas salmonicidae using RPA-CRISPRCas12a, characterized in that... Rapid detection of the crRNA sequence of Aeromonas salmonicidae using RPA-CRISPRCas12a: fstA-crRNA2 is 5'-UAAUUUCUACUAAGUGUAGAUUCGGCAAGCGCUAUGUCACCG-3'.

3. A rapid detection kit for Aeromonas salmonicidae using RPA-CRISPRCas12a, characterized in that, The kit contains the RPA amplification primers as described in claim 1 and the crRNA as described in claim 2.

4. A method for rapid detection of Aeromonas salmonii, characterized in that... The specific steps are as follows: I. Prepare a total RPA reaction system of 50 μL: 29.5 µL of RPA reaction reagent Primer Free Rehydration Buffer, 2.4 µL of primer fstA-RPA-F2 with a concentration of 10 µmol / L, 2.4 µL of primer fstA-RPA-R2 with a concentration of 10 µmol / L, 2.5 µL of magnesium acetate with a concentration of 280 mmol / L, 1 µL of DNA template of the sample to be tested, and the remainder Nuclease-free H2O; II. RPA isothermal amplification: The reaction conditions were 39℃ for 20 min to obtain RPA amplification products; III. RPA-CRISPR / Cas12a reaction: Prepare the CRISPR reaction system and react at 37℃ for 30 min; the CRISPR reaction system consists of: 2 μL Cas12a Reaction buffer 10x, 1 μL probe with a concentration of 5 μmol / L, 1 μL Cas12a protein with a concentration of 1 μmol / L, 1 μL crRNA as described in claim 2 with a concentration of 1 μmol / L, 4 μL RPA amplification product, and 11 μL Nuclease-free H2O; IV. Determine the results by observing the fluorescence curve or the color change of the product in the tube; The probe sequence and fluorescent group are as follows: the fluorescent ssDNA reporter sequence is 5'-FAM-TTTAATTT-BHQ1-3'.

5. The method according to claim 4, characterized in that, Step 1: Preparation of the total RPA reaction system: Add all components except magnesium acetate and DNA template to the reaction tube containing RPA reaction reagent powder, vortex thoroughly, centrifuge, open the reaction tube, add 2.5 μL of magnesium acetate to the cap of the reaction tube, then add the template DNA to the reaction tube, mix thoroughly and centrifuge.

6. A method for rapid detection of Aeromonas salmonidae, characterized in that... The specific steps are as follows: I. Prepare a total RPA reaction system of 50 μL: 29.5 µL of RPA reaction reagent Primer Free Rehydration Buffer, 2.4 µL of primer fstA-RPA-F2 with a concentration of 10 µmol / L, 2.4 µL of primer fstA-RPA-R2 with a concentration of 10 µmol / L, 2.5 µL of magnesium acetate with a concentration of 280 mmol / L, 1 µL of DNA template of the sample to be tested, and the remainder Nuclease-free H2O; II. RPA isothermal amplification: The reaction conditions were 39℃ for 20 min to obtain RPA amplification products; III. CRISPR / Cas12a reaction: Prepare the CRISPR reaction system and react at 37℃ for 30 min; the CRISPR reaction system consists of: 2 μL Cas12a Reaction buffer 10x, 1 μL probe with a concentration of 1 μmol / L, 1 μL Cas12a protein with a concentration of 1 μmol / L, 1 μL crRNA as described in claim 2 with a concentration of 1 μmol / L, 4 μL RPA amplification product, and 11 μL Nuclease-free H2O; 4. Take 10 μL of CRISPR / Cas12a reaction product and add it to a 1.5 mL centrifuge tube containing 40 μL ddH2O. After mixing, put the test strip in and wait 5~10 min to get the test strip detection result. The probe sequence and fluorescent group are as follows: The sequence of the test strip reporter is 5'-FAM-TTTAATTT-Biotin-3'.

7. The method according to claim 6, characterized in that, Step 1: Preparation of the total RPA reaction system: Add all components except magnesium acetate and DNA template to the reaction tube containing RPA reaction reagent powder, vortex thoroughly, centrifuge, open the reaction tube, add 2.5 μL of magnesium acetate to the cap of the reaction tube, then add the template DNA to the reaction tube, mix thoroughly and centrifuge.

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