Primers and applications for rapid detection of pathogenic Aeromonas hydrophila by duplex PCR

Abstract

This invention belongs to the field of microbial detection technology, and relates to primers and their application for rapid detection of pathogenic Aeromonas hydrophila using duplex PCR. This invention is the first to... gyrB and aerA Gene-linked PCR targets were used as dual PCR targets for the detection of Aeromonas hydrophila in artificially infected zebrafish visceral tissue. The positive detection rate was 100% at different infection concentrations. It could distinguish between pathogenic (double bands) and non-pathogenic (only one band). gyrB (Banded) Aeromonas hydrophila. The method of this invention is convenient and rapid, and can complete all detection operations within 1.5 h without the need for expensive instruments and equipment; it has high sensitivity, with a lower limit of detection of nucleic acid of 1 pg / µL and a lower limit of detection of bacterial concentration of 755 CFU / mL; and it has good specificity.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, and relates to primers and their applications for rapid detection of pathogenic Aeromonas hydrophila by dual PCR. Background Technology

[0002] Laboratory fish (such as zebrafish and killifish) are important model organisms in life sciences, medicine, and environmental science. They possess advantages such as short reproductive cycles, transparent embryos, and clear genetic backgrounds, and are widely used in disease model construction, drug screening, and toxicological evaluation. The health status of laboratory fish directly determines the accuracy, reliability, and reproducibility of experimental data and results. Pathogenic Aeromonas hydrophila is one of the core pathogens threatening the safety of laboratory fish farming.

[0003] Aeromonas hydrophila, a Gram-negative pathogen widely found in aquatic and aquaculture environments, frequently infects laboratory fish such as zebrafish and various aquatic animals, inducing septicemia, enteritis, and other diseases. This not only leads to fish mortality and the failure of animal model construction but also interferes with the accuracy and reproducibility of research data, severely hindering the standardization of laboratory fish quality. Currently, detection of this bacterium largely relies on culture and isolation combined with biochemical identification, which is cumbersome and time-consuming, and makes it difficult to distinguish between phenotypic pathogenic and non-pathogenic strains. Single PCR technology can only achieve species identification or pathogenicity screening, but suffers from poor specificity and is prone to missed and false positives, failing to meet the high-efficiency and accurate detection needs in the field of laboratory animals. Therefore, establishing a detection method that can simultaneously complete species identification and pathogenicity assessment is of great significance for the microbial quality monitoring of laboratory fish and the standardization of scientific research experiments.

[0004] Currently, the detection methods for Aeromonas hydrophila in experimental fish mainly follow traditional microbiological detection methods and routine molecular biological detection methods, specifically including:

[0005] 1. Traditional detection methods

[0006] The process, which combines isolation and culture with biochemical identification, includes: isolating and purifying bacterial strains from diseased samples (such as fish tissue, water, and feed), and identifying them through Gram staining, morphological observation, and biochemical indicators; and detecting Aeromonas hydrophila using skim milk plate test and blood plate test. A positive result for either test indicates pathogenic Aeromonas hydrophila (GB / T 18652-2025 Test Methods for Pathogenic Aeromonas hydrophila). This method is a classic approach for microbial detection, with low operating costs, but it has significant limitations: ① Long detection cycle, requiring 3-7 days for the complete process, which cannot meet the needs of early rapid diagnosis and may delay the timing of prevention and control; ② Low sensitivity, when the pathogen content in the sample is low or it is severely contaminated by other bacteria, it is difficult to effectively isolate and purify the target strain, which may lead to false negatives; ③ Insufficient specificity, as the morphological and biochemical characteristics of some closely related species of Aeromonas (such as Aeromonas tempera and Aeromonas guinea pig) are highly similar to those of Aeromonas hydrophila, making it difficult to accurately distinguish them using routine biochemical indicators; ④ The criteria for identifying whether a strain is pathogenic are vague. The "GB / T 18652-2025 Test Method for Pathogenic Aeromonas hydrophila" only specifies that any clear zone in the skim milk plate test and blood plate test is considered positive, but it does not specify the specific diameter of the clear zone, does not provide corresponding solutions for suspicious results, and does not provide standard strains, making inter-laboratory comparison impossible.

[0007] 2. Molecular biological detection methods

[0008] With the development of molecular biology techniques, nucleic acid target-based detection methods have gradually become a research hotspot due to their rapid and sensitive advantages. These mainly include: ① Single-gene PCR detection: GB / T 18652-2025, "Test Methods for Pathogenic Aeromonas hydrophila," uses primers designed with the bacterial conserved gene (gyrB) as the target and achieves detection through conventional PCR amplification. Although gyrB is easier to distinguish between closely related species within the Aeromonas genus than 16S rRNA, a single housekeeping gene can only achieve species-level identification and cannot distinguish whether a strain is pathogenic. ② Double-gene PCR detection: SN / T 4739-2016, "Technical Specifications for Quarantine of Pathogenic Aeromonas hydrophila," selects primers designed with 16S rRNA and the aerA gene as targets and achieves identification of pathogenic Aeromonas hydrophila through two conventional PCR amplifications. However, 16S rRNA is difficult to distinguish between closely related species within the Aeromonas genus, lacks specificity, and is prone to false positives. ③ Duplex PCR detection: Duplex PCR systems suffer from problems such as primer interference and amplification efficiency imbalance, which affect the accuracy and stability of the detection results.

[0009] Deficiencies and shortcomings of existing technologies

[0010] The integration of specificity and pathogenicity identification is insufficient: traditional methods are difficult to distinguish Aeromonas hydrophila from closely related species; single-gene PCR detection can only achieve species identification or can only associate pathogenicity, which cannot simultaneously meet the dual requirements of experimental fish for "precise species identification" and "no pathogenic contamination", which can easily lead to misjudgment or missed detection.

[0011] Poor sample fit and difficulty in matching the characteristics of experimental fish: Experimental fish samples are mostly small amounts of tissue (such as juvenile fish liver), embryos or low concentrations of polluted water. Existing methods are not sensitive enough to detect low levels of pathogens.

[0012] The detection cycle does not match the experimental needs: Traditional methods take 3-7 days, which cannot quickly respond to the emergency detection needs of experimental fish farming, and may delay the timing of pollution control and cause loss of experimental materials; Although the PCR method has a shorter cycle, it is cumbersome to operate and still cannot meet the timeliness requirements of the experimental process.

[0013] In summary, existing detection methods have significant shortcomings in terms of specificity, adaptability, timeliness, and standardization in experimental fish scenarios. There is an urgent need to develop a dual PCR detection method with reasonable target design, high specificity, high sensitivity, simple operation, and adaptability to the characteristics of experimental fish samples to solve the core pain points of detecting pathogenic Aeromonas hydrophila in experimental fish. Summary of the Invention

[0014] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a dual PCR rapid detection method for pathogenic Aeromonas hydrophila in experimental fish, and two pairs of primers for detection.

[0015] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0016] The first aspect of this invention discloses a primer set for rapid detection of pathogenic Aeromonas hydrophila by duplex PCR, the primer set comprising:

[0017] The primer pair targeting the species-specific gene gyrB of Aeromonas hydrophila has the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2;

[0018] GGTCGTGGTATTCCTGTCG (SEQ ID NO:1);

[0019] GCCTTCGTAGCAGAAGTGC (SEQ ID NO:2);

[0020] And primer pairs targeting the pathogenicity-related gene aerA, the nucleotide sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:4;

[0021] GTTATGCCTGGGTGGGTG (SEQ ID NO:3);

[0022] GGGTGTCGCTGTCGTTGAT (SEQ ID NO: 4).

[0023] This invention is the first to combine the species-specific gene gyrB with the pathogenicity-related gene aerA as a dual PCR target for the detection of Aeromonas hydrophila. The target region is highly conserved within Aeromonas hydrophila and differs from closely related species (such as Aeromonas vesiculosus, Aeromonas temperate, and Aeromonas salmonicida) and interfering bacteria (such as Vibrio cholerae and Escherichia coli), thus ensuring the species specificity and pathogenicity differentiation capability of the dual PCR.

[0024] A second aspect of the present invention discloses a kit for rapid detection of pathogenic Aeromonas hydrophila by dual PCR, the kit comprising the primer combination as described in claim 1.

[0025] The third aspect of this invention discloses the application of the above-described primer combinations or kits in the preparation of a method for detecting pathogenic Aeromonas hydrophila in experimental fish.

[0026] The third aspect of this invention discloses a method for rapid detection of pathogenic Aeromonas hydrophila by duplex PCR, comprising the following steps:

[0027] (1) Extract genomic DNA from the sample to be tested;

[0028] (2) Using the genomic DNA extracted in step (1) as a template, a double PCR reaction system was prepared and double PCR amplification was performed using the primer combination described in claim 1;

[0029] (3) Detect the amplification products to determine whether pathogenic Aeromonas hydrophila are present in the sample to be tested.

[0030] The dual PCR reaction system described in step (2) is as follows: In a 25 µL reaction system, there is 1 µL of DNA template, 1 µL each of the primers shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 at a concentration of 10 µM, 12.5 µL of 2×Rapid Taq Plus Master Mix, and the remainder is ddH2O.

[0031] The samples to be tested include genomic DNA of Aeromonas hydrophila, Aeromonas hydrophila colonies, or tissue samples containing Aeromonas hydrophila infection.

[0032] Compared with the prior art, the beneficial effects of this invention are as follows:

[0033] This invention is the first to combine the gyrB and aerA genes as dual PCR targets for the detection of Aeromonas hydrophila. The positive detection rate was 100% in the visceral tissue of artificially infected zebrafish, and the pathogenic (double band) and non-pathogenic (gyrB band only) Aeromonas hydrophila can be distinguished.

[0034] This invention offers a convenient and rapid method that can complete all detection operations within 1.5 hours without requiring expensive instruments or equipment. It exhibits high sensitivity, with a detection limit of 1 pg / µL for nucleic acids and 755 CFU / mL for bacterial concentration. It also demonstrates good specificity; in designing the two primer pairs for the gyrB and aerA gene sequences, the method compares and analyzes the sequences with identical genes from closely related species, selecting nucleotide sequences that are highly consistent in *Aeromonas hydrophila* and non-complementary in other closely related *Aeromonas* species and common interfering species as target sequences, thus demonstrating high specificity. This invention can also be used for the detection and identification of tissue samples infected with *Aeromonas hydrophila*. Furthermore, this invention provides an accurate and efficient detection technique for identifying pathogenic *Aeromonas hydrophila* in experimental fish, offering a theoretical basis for the accurate diagnosis and quarantine of pathogenic *Aeromonas hydrophila*. Attached Figure Description

[0035] Figure 1 This is an agarose gel electrophoresis of the optimized annealing temperature for single-phase PCR of pathogenic Aeromonas hydrophila; where A) is the amplification diagram of the gyrB gene, and B) is the amplification diagram of the aerA gene; M represents the DNA 2000 bp size marker; 1~3 correspond to annealing temperatures of 56℃, 58℃, and 60℃, respectively; N represents the physiological saline negative control.

[0036] Figure 2 This is agarose gel electrophoresis of specific PCR results for pathogenic Aeromonas hydrophila; where M represents a 2000 bp DNA marker; P represents the genomic DNA of the pathogenic Aeromonas hydrophila ATCC7966 reference strain; 1-7 represent Edwardsiella tarda, Aeromonas versicolor, Aeromonas temperate, Aeromonas salmonicida, Shigella-like, Vibrio cholerae, and Escherichia coli, respectively; N represents the saline negative control.

[0037] Figure 3 This is an agarose gel electrophoresis of the optimized annealing temperature for double PCR of pathogenic Aeromonas hydrophila; where M represents a 2000 bp DNA marker; 1-3 correspond to annealing temperatures of 56℃, 58℃, and 60℃, respectively; and N represents a physiological saline negative control.

[0038] Figure 4This is an agarose gel electrophoresis of the optimized final concentration of primers for the duplex PCR of pathogenic Aeromonas hydrophila; where A) is the amplification diagram of primers P-1 and P-2 with a fixed final concentration of 0.4 μmol / L; B) is the amplification diagram of primers P-3 and P-4 with a fixed final concentration of 0.4 μmol / L; M represents the DNA 2000 bp size marker; 1~5 represent the final concentrations of primers P-3 and P-4 as 0.2, 0.3, 0.4, 0.5, and 0.6 μmol / L, respectively; 6~9 represent the final concentrations of primers P-1 and P-2 as 0.2, 0.3, 0.5, and 0.6 μmol / L, respectively; N represents the physiological saline negative control.

[0039] Figure 5 This is agarose gel electrophoresis for specific double PCR of pathogenic Aeromonas hydrophila; where M represents the 2000 bp DNA marker; P represents the genomic DNA of the pathogenic Aeromonas hydrophila ATCC7966 reference strain; 1-7 represent Edwardsiella tarda, Aeromonas versicolor, Aeromonas temperate, Aeromonas salmonicida, Shigella-like, Vibrio cholerae, and Escherichia coli, respectively; N represents the saline negative control.

[0040] Figure 6 This refers to the sensitivity of specific primers to the DNA of pathogenic Aeromonas hydrophila; where M represents a 2000 bp DNA marker; 1-8 represent the concentrations of pathogenic Aeromonas hydrophila ATCC 7966 DNA (1×10⁻⁸). 1 1×10 0 1×10 -1 1×10 -2 1×10 -3 1×10 -4 1×10 -5 1×10 -6 ng / µL; N represents the saline negative control.

[0041] Figure 7 This represents the sensitivity of specific primers to bacterial suspensions of pathogenic Aeromonas hydrophila; where M represents a 2000 bp DNA marker; 1-11 represent bacterial suspension concentrations of pathogenic Aeromonas hydrophila ATCC 7966 (7.55 × 10⁻¹⁰). 6 7.55×10 5 7.55×10 4 7.55×10 3 7.55×10 2 7.55×10 1 7.55×10 0 7.55×10 -1 7.55×10 -2 7.55×10-3 7.55×10 -4 CFU / mL, where N represents the saline negative control.

[0042] Figure 8 This is an agarose gel electrophoresis assay to verify the double PCR of pathogenic Aeromonas hydrophila; where M represents the 2000bp DNA marker; P represents the Aeromonas hydrophila ATCC 7966 standard strain; 1-3 are ZF24012230-1, F2025071523-5, and F2025071523-10, respectively; N represents the physiological saline negative control.

[0043] Figure 9 This is an agarose gel electrophoresis assay using dual PCR to detect pathogenic Aeromonas hydrophila in artificially simulated zebrafish infection; where M represents a 2000 bp DNA marker; P represents the Aeromonas hydrophila ATCC 7966 standard strain; 1-9 are tissue DNA samples from simulated infected tissue samples; and N represents a saline negative control. Detailed Implementation

[0044] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Process parameters not specified in the embodiments of this application can be performed according to conventional methods. Experimental methods without specific experimental conditions are generally performed according to conventional experimental conditions or the experimental conditions quarantined by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0045] The main reagents and instruments used in this invention are:

[0046] Bacterial DNA Extraction Kit (Beijing Jinsha Biotechnology Co., Ltd.), Rapid Tissue DNA Extraction Kit (Onobate Biotechnology Co., Ltd.), 2×Rapid Taq Plus Master Mix (Novizan Biotechnology Co., Ltd.), Nucleic Acid Dyes and DL 2000 Marker (Sangon Biotech (Shanghai) Co., Ltd.), Columbia Blood Plates, 1% Skim Milk Plates (Shanghai Komage Biotechnology Co., Ltd.), FlaPure Gel Purification Kit DNA Gel Recovery Kit (Beijing Jinsha Biotechnology Co., Ltd.); Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. PCR instrument (Thermo Fisher Scientific), gel imaging system (Bio-Rad, USA), nucleic acid electrophoresis instrument (Bio-Rad, USA), NanoDrop 1000 microspectrophotometer (Nanodrop, USA).

[0047] The Aeromonas hydrophila ATCC 7966 and Edwardsiella tarda ATCC 15947 used in the examples were purchased and preserved by our laboratory (Shanghai Fuxiang Biotechnology Co., Ltd.). Pathogenic Aeromonas hydrophila (ZF24012230-1), non-pathogenic Aeromonas hydrophila (F2025071523-5, F2025071523-10), Aeromonas verrucosa (240124-7), Aeromonas temperate (231030-26), Aeromonas salmonicida (240315-4), Shigella-like (231030-29), Vibrio cholerae (250424-22), and Escherichia coli (241212-1) were isolated and preserved by our laboratory.

[0048] Example 1

[0049] The establishment of a single-phase PCR system includes the following steps:

[0050] (1) Template preparation: Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium marinum, Aeromonas versicolor, Aeromonas vesiculosus, Aeromonas sobrio, Aeromonas salmonicidae, Shigella shigella, Vibrio cholerae and Escherichia coli were picked from the bacterial culture tubes stored at -80℃ and inoculated onto Columbia blood agar plates under aseptic conditions. The culture was carried out at 28℃ for 24 h. After that, single colonies were picked and purified for 16 h. Single colonies were picked onto brain heart infusion agar medium and cultured at 28℃ for 16 h at a shaking speed of 170 rpm. Genomic DNA was extracted using a DNA extraction kit and used as template.

[0051] (2) Primer design and synthesis: The complete sequences of gyrB and aerA from the whole genome of Aeromonas hydrophila ATCC 7966 in GenBank and the corresponding sequences of other common aquatic pathogens were retrieved. Sequence alignment was performed using SerialCloner. Nucleotide sequences that are highly consistent in Aeromonas hydrophila and non-complementary in other closely related species of Aeromonas and common interfering species were selected as target sequences to design primers P-1, P-2, P-3, and P-4. The expected target fragment size of the gyrB gene is 435 bp, and the expected target fragment size of the aerA gene is 261 bp.

[0052] (3) The primer sequences are as follows:

[0053] PCR primers targeting the gyrB gene of pathogenic Aeromonas hydrophila:

[0054] P-1: 5'-GGTCGTGGTATTCCTGTCG-3';

[0055] P-2: 5'-GCCTTCGTAGCAGAAGTGC-3';

[0056] PCR primers targeting the aerA gene of pathogenic Aeromonas hydrophila:

[0057] P-3: 5'-GTTATGCCTGGGTGGGTG -3';

[0058] P-4: 5'- GGGTGTCGCTGTCGTTGAT-3'.

[0059] (4) Optimization of annealing temperature for single PCR primers: The 25 µL reaction mixture contained 1 µL of Aeromonas hydrophila ATCC 7966 DNA, 0.75 µL each of primers P-1, P-2, P-3, and P-4 (primer concentration 10 µM), 12.5 µL of 2×Rapid Taq Plus Master Mix, and ddH2O was added to bring the total to 25 µL. The reaction program was as follows: 95℃ pre-denaturation for 2 min; 30 cycles of 98℃ for 10 sec, 56, 58, and 60℃ for 30 sec, and 68℃ for 15 sec; and a final extension at 68℃ for 5 min to explore the optimal annealing temperature for the two primer pairs. The 1.5% agarose gel electrophoresis results are shown below. Figure 1 The optimal annealing temperature for the two primer pairs is 58℃.

[0060] (5) Specificity of single PCR: Using the amplification system with the optimal annealing temperature selected above, DNA of Aeromonas hydrophila ATCC 7966 and seven non-target bacteria (Aeromonas velutipes, Aeromonas sobrio, Aeromonas salmonicida, Shigella-like bacteria, Vibrio cholerae, Edwardsiella tarda, and Escherichia coli) were amplified. The DNA of Aeromonas hydrophila ATCC 7966 was used as a positive control, and physiological saline as a negative control to determine whether the two primer pairs showed cross-reactivity with other bacteria. The results of 1.5% agarose gel electrophoresis are shown below. Figure 2 Using Aeromonas hydrophila as a template, the corresponding band was amplified, while no bands were amplified for other strains, indicating that the two primer pairs had good specificity.

[0061] Example 2

[0062] The establishment of a rapid doublet PCR method for detecting pathogenic Aeromonas hydrophila includes the following steps:

[0063] (1) Optimization of annealing temperature for duplex PCR: The reaction system was set up as follows: 25 µL of reaction mixture contained 1 µL of Aeromonas hydrophila ATCC 7966 DNA, 0.75 µL each of primers P-1, P-2, P-3 and P-4 (primer concentration 10 µM), 12.5 µL of 2×Rapid Taq Plus Master Mix, and ddH2O was added to 25 µL; the reaction program was as follows: 95℃ pre-denaturation for 2 min; 30 cycles of 98℃ for 10 sec, 58, 56, 54℃ for 30 sec and 68℃ for 15 sec; and a final extension at 68℃ for 5 min to explore the optimal annealing temperature for duplex PCR. The results of 1.5% agarose gel electrophoresis are shown below. Figure 3 When the annealing temperature is 58℃, double PCR can amplify two clear bands, which are consistent with the expected results of 435 bp and 261 bp band positions.

[0064] (2) Optimization of final primer concentrations for duplex PCR: Using the final concentrations of the two primer pairs in the duplex PCR reaction system as variables, a gradient combination experiment was designed using the "single-factor fixation method" to screen the optimal primer final concentration ratio in two groups, ensuring the optimization was targeted and accurate. One group fixed the final concentration of gyrB gene primers (P-1, P-2) at 0.4 μmol / L, and only adjusted the final concentration of aerA gene primers (P-3, P-4), setting 5 gradients of 0.2, 0.3, 0.4, 0.5, and 0.6 μmol / L; the other group fixed the final concentration of aerA gene primers (P-3, P-4) at 0.4 μmol / L, and only adjusted the final concentration of gyrB gene primers (P-1, P-2), setting 4 gradients of 0.2, 0.3, 0.5, and 0.6 μmol / L, forming a total of 9 concentration combinations. The reaction mixture consisted of 1 µL of Aeromonas hydrophila ATCC 7966 DNA in a 25 µL reaction system. Primers P-1, P-2, P-3, and P-4 (primer concentration 10 µM) were added, with the addition volume adjusted according to the final concentration. 12.5 µL of 2×Rapid Taq Plus Master Mix was added, and ddH2O was added to bring the total to 25 µL. The reaction program was: 95℃ pre-denaturation for 2 min; 30 cycles of 98℃ for 10 sec, 58℃ for 30 sec, and 68℃ for 15 sec; and a final extension at 68℃ for 5 min to explore the optimal final primer concentration for duplex PCR. 1.5% agarose gel electrophoresis results are shown below. Figure 4 When the final concentrations of P-1 and P-2 were 0.4 μmol / L, the target band of the aerA gene gradually brightened with increasing final concentration, but the brightness of the target band did not change significantly at concentrations of 0.4, 0.5, and 0.6 μmol / L. Figure 5(A) When the final concentrations of P-3 and P-4 are 0.4 μmol / L, and the final concentrations of P-1 and P-2 are 0.5 μmol / L, the corresponding two target bands are the brightest. Figure 5 (B); In summary, 0.4 μmol / L was selected as the optimal final concentration of the two primer pairs for the subsequent reaction system.

[0065] (3) Specificity detection by duplex PCR: The DNA of *Aeromonas hydrophila* ATCC 7966 and seven non-target bacteria (*Aeromonas versicolor*, *Aeromonas sobria*, *Aeromonas salmonicida*, *Shigella*, *Vibrio cholerae*, *Edwards tarda*, and *Escherichia coli*) was used as templates to test the specificity of the duplex PCR technique. The results of 1.5% agarose gel electrophoresis are shown below. Figure 5 Two target bands were amplified from the genomic DNA sample of Aeromonas hydrophila ATCC7966, with lengths of 435 bp and 261 bp, respectively; however, no bands were observed when amplifying the DNA templates of seven non-target bacteria.

[0066] (4) Based on the results of the above primer final concentration optimization experiment, combined with the gradient exploration of key parameters such as annealing temperature, and through comprehensive evaluation of amplification specificity and band brightness, the optimal reaction system and conditions for the dual PCR detection of pathogenic Aeromonas hydrophila were finally established: 25 µL of reaction system mixture is 1 µL of template, primers P-1, P-2, P-3 and P-4 (primer concentration of 10 µM) are 1 µL each, 12.5 µL of 2×Rapid Taq Plus Master Mix, and ddH2O is added to 25 µL; reaction program: 95℃ pre-denaturation for 2 min; 30 cycles of 98℃ for 10 sec, 58℃ for 30 sec and 68℃ for 15 sec; final extension at 68℃ for 5 min; electrophoresis conditions are 1.5% agarose gel, 1×TAE buffer, 100 V constant voltage electrophoresis for 30 min.

[0067] Example 3

[0068] The sensitivity of double PCR detection for pathogenic Aeromonas hydrophila DNA includes the following steps:

[0069] (1) The concentration of DNA of Aeromonas hydrophila ATCC 7966 extracted in step (1) of Example 1 was determined using a spectrophotometer;

[0070] (2) Dilute the genomic DNA of Aeromonas hydrophila to 1×10⁻⁶. 1 1×10 0 1×10 -1 1×10 -2 1×10 -3 1×10 -4 1×10-5 ng / µL, using the reaction system, reaction procedure and electrophoresis conditions in step (4) of Example 2 for double PCR.

[0071] (3) such as Figure 6 As shown, the DNA sensitivity of pathogenic Aeromonas hydrophila is 1 pg / µL.

[0072] Example 4

[0073] The sensitivity of double PCR detection of Aeromonas hydrophila bacterial suspensions includes the following steps:

[0074] (1) Take activated Aeromonas hydrophila ATCC 7966 bacterial suspension, wash twice with 0.9% physiological saline, dilute in a 10-fold gradient, and plate for viable count;

[0075] (2) The bacterial concentration of Aeromonas hydrophila was diluted to 7.55 × 10⁻⁶. 6 7.55×10 5 7.55×10 4 7.55×10 3 7.55×10 2 7.55×10 1 7.55×10 0 7.55×10 -1 7.55×10 -2 7.55×10 -3 7.55×10 -4 CFU / mL.

[0076] (3) Genomic DNA was extracted from the bacterial culture using a DNA extraction kit, and double PCR was performed using the reaction system, reaction procedure and electrophoresis conditions in step (9) of Example 1.

[0077] (4) such as Figure 7 As shown, the bacterial suspension sensitivity of pathogenic Aeromonas hydrophila is 755 CFU / mL.

[0078] Example 5

[0079] The reproducibility and stability of the doublet PCR method for detecting pathogenic Aeromonas hydrophila include the following steps:

[0080] (1) Use the same batch of Aeromonas hydrophila ATCC 7966 DNA template prepared and stored at -20℃ (avoid repeated freeze-thaw cycles). Primers, 2×Rapid Taq Plus Master Mix and other reagents should also be from the same batch to ensure reagent consistency.

[0081] (2) Three parallel reaction systems were prepared independently on two PCR instruments, and then amplified according to the established procedure. After amplification, all samples were tested under the same conditions of 1.5% agarose gel, 1×TAE buffer, and constant voltage electrophoresis at 100 V for 30 min. The same gel imaging system was used to take pictures and record the results. The above operations were carried out once a month at a fixed time for 5 consecutive months, covering different environmental temperature and humidity conditions to simulate long-term routine testing scenarios and to verify the repeatability and stability of the method.

[0082] (3) As shown in Table 1, using the DNA template of Aeromonas hydrophila ATCC 7966 prepared in the same batch, primers, 2×Rapid Taq Plus Master Mix and other reagents, PCR amplification was carried out on two PCR instruments at fixed times every month for 5 consecutive months. Specific bands appeared at the corresponding size in all cases, indicating that the dual PCR detection method has good repeatability and stability.

[0083] Table 1. Results of duplex PCR repeatability and stability

[0084] Example 6

[0085] Validation of the doublet PCR method for detecting pathogenic Aeromonas hydrophila includes the following steps:

[0086] (1) Duplex PCR was used to detect pathogenic Aeromonas hydrophila (ZF24012230-1) and non-pathogenic Aeromonas hydrophila (F2025071523-5, F2025071523-10) previously identified by traditional methods. Using extracted pathogenic and non-pathogenic Aeromonas hydrophila DNA as templates, the amplification system and conditions were followed. The amplified products were detected by 1.5% agarose gel electrophoresis. After confirming that the target band size was consistent with expectations, the target band was excised and purified using gel electrophoresis. The purified PCR products were sent to a sequencing company for Sanger sequencing using bidirectional sequencing. The sequencing results were compared with known Aeromonas hydrophila sequences in the GenBank database using the NCBI BLAST tool to verify the accuracy of the amplified products and ensure the reliability of the strain identification results.

[0087] (2) For example Figure 8 As shown, ZF24012230-1, which has a clear ring as verified by clinical 1% skim milk plate, amplified two specific bands simultaneously. F2025071523-5 and F2025071523-10, which do not have a clear ring as verified by clinical 1% skim milk plate, only amplified the target fragment of the gyrB gene, which is consistent with the traditional identification results.

[0088] Example 7: Application of duplex PCR detection of pathogenic Aeromonas hydrophila in artificially simulated zebrafish infection, including the following steps:

[0089] (1) Under aseptic conditions, dissect four healthy zebrafish and take visceral tissue (150 mg). Place each tissue in a sterile grinding tube and add 500 μL of sterile PBS buffer. Homogenize on ice until homogenized to prepare four stock solutions of healthy tissue homogenate. Divide each of the four stock solutions of tissue homogenate into three replicates (200 μL per replicate, placed in a 1.5 mL sterile centrifuge tube) as experimental groups. Add 20 μL of Aeromonas hydrophila bacterial suspension of different concentrations (1×10⁻⁶) to each of the three replicates of tissue homogenate in the experimental groups. 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 (CFU / mL), and for the blank control group, 20 μL of sterile saline was added to each of the three replicates of tissue homogenate. The mixture was gently vortexed for 30 s to ensure thorough mixing of the bacterial solution and tissue homogenate. The mixture was then incubated at 28°C for 1 h. The prepared simulated infected tissue sample (200 μL, containing the mixture of tissue homogenate and bacterial solution) was then placed in a 1.5 mL sterile centrifuge tube.

[0090] (2) Follow the instructions of the tissue genomic DNA extraction kit, and then amplify according to the established procedure. After the amplification is completed, all samples are tested under the conditions of 1.5% agarose gel, 1×TAE buffer, 100 V constant voltage electrophoresis for 30 min.

[0091] (3) such as Figure 9 We used double PCR to amplify the nucleic acid samples of zebrafish viscera that were artificially infected. We found that different infection concentrations could simultaneously amplify specific bands of 435 bp and 261 bp, with a positive detection rate of 100%.

[0092] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.

Claims

1. A primer combination for rapid detection of pathogenic Aeromonas hydrophila by duplex PCR, characterized in that, The primer combination includes: Species-specific genes of Aeromonas hydrophila gyrB The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:2; and pathogenicity-related genes aerA The primer pairs, whose nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:

4.

2. A kit for rapid detection of pathogenic Aeromonas hydrophila using dual PCR, characterized in that, The kit comprises the primer combination as described in claim 1.

3. The application of the primer combination as described in claim 1 or the kit as described in claim 2 in the preparation of a kit for detecting pathogenic Aeromonas hydrophila in experimental fish.

4. A method for rapid detection of pathogenic Aeromonas hydrophila by duplex PCR, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, a double PCR reaction system was prepared and double PCR amplification was performed using the primer combination described in claim 1; (3) Detect the amplification products to determine whether pathogenic Aeromonas hydrophila are present in the sample to be tested.

5. The method according to claim 4, characterized in that, The dual PCR reaction system described in step (2) is as follows: in a 25µL reaction system, there is 1 µL of DNA template, 1 µL each of the primers shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 at a concentration of 10 µM, 12.5 µL of 2×Rapid Taq Plus Master Mix, and the remainder is ddH2O.

6. The method according to claim 4, characterized in that, The samples to be tested include genomic DNA of Aeromonas hydrophila, Aeromonas hydrophila colonies, or tissue samples containing Aeromonas hydrophila infection.

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