Multiple qPCR (quantitative polymerase chain reaction) primer group and kit for detecting core pathogenic bacteria in aquaculture and application of multiple qPCR primer group and kit

Abstract

The invention discloses multiple qPCR (quantitative polymerase chain reaction) primers and a kit for detecting core pathogenic bacteria in aquaculture and application of the multiple qPCR primers and the kit. The multiple qPCR primer group comprises a primer combination A (SEQ ID NO.1-3) for detecting edwardsiella tarda, a primer combination B (SEQ ID NO.4-6) for detecting streptococcus iniae and a primer combination C (SEQ ID NO.7-9) for detecting aeromonas salmonicida. The invention also provides a kit containing the primer group and a detection method. A single-tube multiple detection system is constructed for three pathogenic bacteria which are most harmful in aquaculture, detection is rapid, and compared with a traditional culture method, the period is greatly shortened; the method is high in flux, low in cost and suitable for large-scale monitoring; and the kit has strong specificity and high sensitivity, and can realize early screening. The invention provides an effective tool for rapid, accurate and high-throughput detection of core pathogenic bacteria in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a multiplex qPCR primer set, kit, and its application for detecting core pathogenic bacteria in aquaculture. Background Technology

[0002] With the increasing intensification of aquaculture, the frequency and severity of bacterial disease outbreaks are escalating, becoming one of the main bottlenecks restricting the sustainable development of the industry. Among these, Edwardsiella tarda (…) Edwardsiella tarda ), Dolphin Streptococcus ( Streptococcus iniae ) and Aeromonas salmonidae ( Aeromonas salmonicida These three pathogens are recognized globally as the most serious threats to aquaculture. Edwardsiella tarda causes Edwardsiella pneumoniae in fish, Streptococcus dolphinii is the primary pathogen of streptococcal disease, and Aeromonas salmonidus causes furunculosis. All three diseases are characterized by insidious infection, short incubation periods, high susceptibility to outbreaks, and high mortality rates. They can infect a variety of important aquaculture species, including salmon, trout, tilapia, and perch, causing enormous economic losses to the global aquaculture industry.

[0003] Accurate and rapid identification of pathogens is a prerequisite for effective disease control and minimizing economic losses. Currently, routine detection methods for these three pathogens mainly rely on traditional bacterial isolation and culture followed by biochemical identification. This method typically requires inoculating pathogen samples into a culture medium, culturing for 24-48 hours to obtain single bacterial colonies, and then performing a series of time-consuming biochemical tests. The entire process often takes 3-7 days to obtain the final identification result. This detection cycle is too long and cannot meet the urgent need for rapid diagnosis and timely control measures in the early stages of a disease outbreak, often missing the optimal control opportunity and leading to the rapid spread of the disease.

[0004] With the development of molecular biology techniques, polymerase chain reaction (PCR)-based detection technologies, such as conventional PCR and real-time quantitative PCR (qPCR), have been increasingly applied to the detection of pathogenic microorganisms due to their advantages such as high sensitivity, strong specificity, and fast detection speed. However, most existing PCR detection methods for Edwardsiella tarda, Streptococcus dolphinii, and Aeromonas salmonicida are single-particle PCR, meaning that a single PCR reaction can only detect one pathogen. In actual aquaculture production, pathogen infections are often very complex, and mixed infections of multiple pathogens frequently occur. Using single-particle PCR requires multiple reactions on the same sample, which is not only cumbersome, has low throughput, consumes a lot of reagents, and is costly, but more importantly, it cannot provide information on the abundance of each pathogen in a mixed infection, making it difficult to comprehensively and accurately assess the epidemic situation, thus affecting the formulation of precise medication and treatment plans.

[0005] Therefore, developing a multiplex detection technology that can simultaneously, rapidly, and accurately detect three core pathogenic bacteria in aquaculture—Edwards tarda, Streptococcus dolphinus, and Aeromonas salmonidae—is of significant practical importance and application value for improving early warning and control capabilities for aquaculture diseases and reducing economic losses. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a multiplex qPCR primer set, kit, and its application for detecting core pathogenic bacteria in aquaculture.

[0007] This invention is achieved through the following technical solution: A multiplex qPCR primer set for detecting core pathogenic bacteria in aquaculture, the multiplex qPCR primer set including primers for detecting Edwardsiella tarda (… Edwardsiella tarda Primer combination A for detecting Streptococcus dolphinus ( Streptococcus iniae Primer combination B for detecting Aeromonas salmonicida ( ) and Aeromonas salmonicida ( ) Aeromonas salmonicida Primer combination C; where, The detection of Edwardsiella tarda ( Edwardsiella tarda Primer combination A includes: Upstream primer: 5'-GGGGTAAGTTCGACGACAAC-3' (SEQ ID NO.1) Downstream primer: 5'-GCGGATCACCAGTTCCAAC-3' (SEQ ID NO.2) Probe: 5'-CCTACAAGGTTTCCGGTGGTCTGC-3' (SEQ ID NO.3), the 5' end of the probe is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the fluorescent quencher group BHQ1; The detection of Streptococcus dolphinus ( Streptococcus iniae Primer combination B includes: Upstream primer: 5'-TCCAGCTAACAACGGTCAA-3' (SEQ ID NO.4) Downstream primer: 5'-CTTCCTCTTTCGCTTTACGG-3' (SEQ ID NO.5) Probe: 5'-AGAGTACCAGCTCCAACCATAACCGC-3' (SEQ ID NO.6), the 5' end of the probe is labeled with a fluorescent reporter group VIC, and the 3' end is labeled with a fluorescent quencher group BHQ1; The detection of Aeromonas salmonicida ( Aeromonas salmonicida The primer combination C includes: Upstream primer: 5'-ACTGTCTGTTACCCTGCCAAG-3' (SEQ ID NO.7) Downstream primer: 5'-CAGCAGTGATAGAAGCACCAAC-3' (SEQ ID NO.8) Probe: 5'-CAGCAGCAACATCAGCAGGCTTCA-3' (SEQ ID NO.9), the 5' end of the probe is labeled with the fluorescent reporter group ROX, and the 3' end is labeled with the fluorescent quencher group BHQ2.

[0008] This invention also provides the application of the above-mentioned multiplex qPCR primer set in the preparation of a kit for the simultaneous detection of Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonicida.

[0009] The present invention also provides a kit for detecting core pathogenic bacteria in aquaculture, the kit being able to simultaneously detect Edwardsiella tarda, Streptococcus dolphinus and Aeromonas salmonicida, the kit comprising the above-mentioned multiplex qPCR primer set; Preferably, the kit further includes: (a) qPCR reaction system premix: The premix contains DNA polymerase, dNTPs and PCR buffer; (b) Positive control: The positive control is a recombinant plasmid containing target gene fragments of Edwardsiella tarda, Streptococcus dolphinus and Aeromonas salmonicidae; (c) Negative control: The negative control is nuclease-free water.

[0010] This invention also provides a method for detecting core pathogenic bacteria in aquaculture using the above-mentioned multiplex qPCR primer set, comprising the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted DNA as a template, multiplex qPCR amplification was performed using a set of multiplex qPCR primers; (3) Collect fluorescence signals, and determine the results based on the amplification curve and Ct value as follows: a. The positive / negative status of the FAM channel: Positive: When the FAM channel amplification result of the sample to be tested shows a typical amplification curve and Ct≤35, it is determined that the sample to be tested contains Edwardsiella tarda. Negative: If the FAM channel amplification result of the sample to be tested has no amplification curve, or if the FAM channel amplification result of the sample to be tested has an amplification curve but Ct≥38, then it is determined that the sample to be tested does not contain Edwardsiella tarda. Suspicious: When an amplification curve appears in the FAM channel amplification result of the test sample and 35 < Ct < 38, it is determined that the test sample is a suspicious sample, and DNA needs to be re-extracted for retesting. b. Positive or negative situation of the VIC channel: Positive: When a typical amplification curve appears in the VIC channel amplification result of the test sample and Ct ≤ 35, it is determined that the test sample contains Streptococcus iniae. Negative: When there is no amplification curve in the VIC channel amplification result of the test sample, or although there is an amplification curve in the VIC channel amplification result of the test sample but Ct ≥ 38, it is determined that the test sample does not contain Streptococcus iniae. Suspicious: When an amplification curve appears in the VIC channel amplification result of the test sample and 35 < Ct < 38, it is determined that the test sample is a suspicious sample, and DNA needs to be re-extracted for retesting.

[0011] c. Positive or negative situation of the ROX channel: Positive: When a typical amplification curve appears in the ROX channel amplification result of the test sample and Ct ≤ 35, it is determined that the test sample contains Aeromonas salmonicida. Negative: When there is no amplification curve in the ROX channel amplification result of the test sample, or although there is an amplification curve in the ROX channel amplification result of the test sample but Ct ≥ 38, it is determined that the test sample does not contain Aeromonas salmonicida. Suspicious: When an amplification curve appears in the ROX channel amplification result of the test sample and 35 < Ct < 38, it is determined that the test sample is a suspicious sample, and DNA needs to be re-extracted for retesting. [[ID=******************]]Beneficial effects of the present invention:

[0012] 1. Innovation of target and scenario: The present invention constructs a multiplex real-time fluorescence quantitative PCR system for simultaneously detecting three core pathogenic bacteria, Edwardsiella tarda, Streptococcus iniae, and Aeromonas salmonicida, which are the most harmful in current aquaculture production. Different from the prior art that mainly focuses on the detection of food processing terminals or human foodborne pathogenic bacteria, the present invention directly serves the active prevention and control of diseases in the aquaculture process, forming a fundamental distinction in the selection of target bacteria and application scenarios, and having prominent novelty and creativity. [[ID=******************]] [[ID=******************]]

[0013] 2. Fast detection speed and high efficiency: The detection method provided by the present invention shortens the time from sample processing to result issuance to 2.5 - 3 hours. Compared with the traditional bacterial isolation culture and biochemical identification methods (usually taking 3 - 7 days), the detection cycle is significantly shortened, which can meet the urgent needs of rapid diagnosis and timely isolation or treatment measures in the early stage of disease outbreaks in aquaculture clinical practice, and effectively avoid the spread of the epidemic.

[0014] 3. High throughput and low detection cost: This invention employs multiplex qPCR technology, requiring only a single-tube reaction to simultaneously detect and identify three target pathogens. Compared to existing technologies that require three singlex PCR reactions to cover all three pathogens, this invention significantly reduces reagent consumption and lowers manual labor and time costs for testing personnel. It is particularly suitable for large-scale epidemiological surveys and routine monitoring in aquaculture enterprises and grassroots testing institutions.

[0015] 4. High Specificity and Accuracy: This invention designs primers and TaqMan probes based on conserved specific target genes of Edwardsiella tarda, Streptococcus dolphinii, and Aeromonas salmonidae. Experimental verification shows that the primers and probes do not cross-react with other common non-target pathogens in aquaculture environments (such as Vibrio, other species of Aeromonas), enabling precise identification of target pathogens and effectively avoiding false positive results caused by non-specific amplification. The detection results are accurate and reliable.

[0016] 5. High Detection Sensitivity: The detection system provided by this invention has high sensitivity, with a detection limit as low as 10 CFU / reaction when detecting pure cultures. This high sensitivity enables the method to achieve early screening and warning of pathogens in the early stages of infection, before the host shows obvious clinical symptoms. This provides valuable time for timely intervention, helps reduce morbidity and mortality, and minimizes economic losses. Attached Figure Description

[0017] Figure 1 Amplification curve for specificity verification experiment; In the figure, samples 1-10 correspond to Edwardsiella tarda, Streptococcus dolphinus, Aeromonas salmonidae, Edwardsiella catfishidae, Streptococcus agalactiae, Aeromonas hydrophila, Escherichia coli, Vibrio anguillarum, Salmonella, and Staphylococcus aureus, respectively. Sample 11 is the negative control.

[0018] Figure 2 This is a standard curve for the multiplex qPCR of this invention.

[0019] Figure 3 10 6 ~10 1 PCR amplification curves using standard templates (copies / μL); The NTC in the figure is a negative control (template: nuclease-free water).

[0020] Figure 4 These are the results of the sensitivity test. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0022] Example 1: Design and Synthesis of Multiplex qPCR Primers for Detecting Core Pathogenic Bacteria in Aquaculture Based on the conserved sequences of the *Edwardsiella tarda* gyrB gene (target 1), *Streptococcus dolphinus* simA gene (target 2), and *Aeromonas salmonicida* VapA gene (target 3) published in GenBank, multiple primer sets and TaqMan probes were designed using primer design software. Through screening and optimization, the final primer set was determined to include those for detecting *Edwardsiella tarda* (…). Edwardsiella tarda Primer combination A for detecting Streptococcus dolphinus ( Streptococcus iniae Primer combination B for detecting Aeromonas salmonicida ( ) and Aeromonas salmonicida ( ) Aeromonas salmonicida Primer combination C; where, The detection of Edwardsiella tarda ( Edwardsiella tarda Primer combination A includes: Upstream primer: 5'-GGGGTAAGTTCGACGACAAC-3' (SEQ ID NO.1) Downstream primer: 5'-GCGGATCACCAGTTCCAAC-3' (SEQ ID NO.2) Probe: 5'-CCTACAAGGTTTCCGGTGGTCTGC-3' (SEQ ID NO.3), the 5' end of the probe is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the fluorescent quencher group BHQ1; The detection of Streptococcus dolphinus ( Streptococcus iniae Primer combination B includes: Upstream primer: 5'-TCCAGCTAACAACGGTCAA-3' (SEQ ID NO.4) Downstream primer: 5'-CTTCCTCTTTCGCTTTACGG-3' (SEQ ID NO.5) Probe: 5'-AGAGTACCAGCTCCAACCATAACCGC-3' (SEQ ID NO.6), the 5' end of the probe is labeled with a fluorescent reporter group VIC, and the 3' end is labeled with a fluorescent quencher group BHQ1; The detection of Aeromonas salmonicida ( Aeromonas salmonicida The primer combination C includes: Upstream primer: 5'-ACTGTCTGTTACCCTGCCAAG-3' (SEQ ID NO.7) Downstream primer: 5'-CAGCAGTGATAGAAGCACCAAC-3' (SEQ ID NO.8) Probe: 5'-CAGCAGCAACATCAGCAGGCTTCA-3' (SEQ ID NO.9), the 5' end of the probe is labeled with the fluorescent reporter group ROX, and the 3' end is labeled with the fluorescent quencher group BHQ2.

[0023] Example 2: Establishment of a multiplex qPCR detection method Using genomic DNA from Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonidae as mixed templates, the multiplex qPCR reaction system and conditions were optimized. The optimal reaction system was determined as follows: 10 μL of 2× multiplex qPCR premix (Hunan Aikerui Biotechnology Co., Ltd., 2X Premixed Probe qPCR Kit II (with UNG), Catalog No. CM0213) 0.4 μL of the upstream primer (10 μM) shown in SEQ ID NO.1 0.4 μL of the upstream primer (10 μM) shown in SEQ ID NO.4 0.4 μL of the upstream primer (10 μM) shown in SEQ ID NO.7 0.4 μL of the downstream primer (10 μM) shown in SEQ ID NO.2 0.4 μL of the downstream primer (10 μM) shown in SEQ ID NO.5 0.4 μL of the downstream primer (10 μM) shown in SEQ ID NO.8 0.2 μL of the probe (10 μM) shown in SEQ ID NO.3 0.2 μL of the probe (10 μM) shown in SEQ ID NO.6 0.2 μL of the probe (10 μM) shown in SEQ ID NO.9 DNA template (mixture of DNA from three pathogens) 2 μL Nuclease-free water was added to a final volume of 20 μL.

[0024] Reaction procedure: 95℃ pre-denaturation for 30 seconds, 95℃ denaturation for 10 seconds, 60℃ annealing and extension for 30 seconds (fluorescence was collected during this stage), for a total of 40 cycles.

[0025] Result interpretation: After the reaction is completed, the results are determined according to the amplification curves and Ct values of the fluorescence signals in each channel (the FAM channel corresponds to *Edwardsiella tarda*, the VIC channel corresponds to *Streptococcus iniae*, and the ROX channel corresponds to *Aeromonas salmonicida*): a. Positive or negative situation of the FAM channel: Positive: When the amplification result of the FAM channel of the test sample shows a typical amplification curve and Ct ≤ 35, it is determined that the test sample contains *Edwardsiella tarda*; Negative: When the amplification result of the FAM channel of the test sample has no amplification curve, or although the amplification result of the FAM channel of the test sample has an amplification curve but Ct ≥ 38, it is determined that the test sample does not contain *Edwardsiella tarda*; Suspicious: When the amplification result of the FAM channel of the test sample shows an amplification curve and 35 < Ct < 38, it is determined that the test sample is a suspicious sample and needs to be retested after re-extracting DNA; b. Positive or negative situation of the VIC channel: Positive: When the amplification result of the VIC channel of the test sample shows a typical amplification curve and Ct ≤ 35, it is determined that the test sample contains *Streptococcus iniae*; Negative: When the amplification result of the VIC channel of the test sample has no amplification curve, or although the amplification result of the VIC channel of the test sample has an amplification curve but Ct ≥ 38, it is determined that the test sample does not contain *Streptococcus iniae*; Suspicious: When the amplification result of the VIC channel of the test sample shows an amplification curve and 35 < Ct < 38, it is determined that the test sample is a suspicious sample and needs to be retested after re-extracting DNA.

[0026] c. Positive or negative situation of the ROX channel: Positive: When the amplification result of the ROX channel of the test sample shows a typical amplification curve and Ct ≤ 35, it is determined that the test sample contains *Aeromonas salmonicida*; Negative: When the amplification result of the ROX channel of the test sample has no amplification curve, or although the amplification result of the ROX channel of the test sample has an amplification curve but Ct ≥ 38, it is determined that the test sample does not contain *Aeromonas salmonicida*; Suspicious: When the amplification result of the ROX channel of the test sample shows an amplification curve and 35 < Ct < 38, it is determined that the test sample is a suspicious sample and needs to be retested after re-extracting DNA.

[0027] Example 3 Specificity verification To evaluate the specificity of the multiplex qPCR detection method established in this invention, using the reaction system and conditions optimized and determined in Example 2, three target pathogenic bacteria, namely *Edwardsiella tarda*, *Streptococcus iniae*, and *Aeromonas salmonicida*, and 7 non-target control strains such as *Edwardsiella ictaluri*, *Streptococcus agalactiae*, and *Aeromonas hydrophila* were detected.

[0028] 1. Experimental strain Target strains: Edwardsiella tarda, Streptococcus dolphinus, Aeromonas salmonidae standard strain; Non-target control strains: Edwardsiella tarda, Streptococcus agalactiae, Aeromonas hydrophila, Escherichia coli, Vibrio anguillarum, Salmonella, Staphylococcus aureus; 2. Detection Method Genomic DNA was extracted from each strain as a template and tested using the multiplex qPCR system established in Example 2, with nuclease-free water as a negative control.

[0029] 3. Experimental Results The results are as follows Figure 1 As shown, the experimental results indicate that, (1) In the FAM detection channel, the reaction wells containing only Edwardsiella tarda template showed a typical S-shaped amplification curve with a Ct value of 23.55. (2) In the VIC detection channel, the reaction wells containing only the Streptococcus dolphin template showed a typical S-shaped amplification curve with a Ct value of 21.17. (3) In the ROX detection channel, the reaction wells containing only Aeromonas salmonidae template showed a typical S-type amplification curve with a Ct value of 18.11. (4) No amplification signal was detected in any of the non-target control strains and negative controls in the three detection channels; The above results show that the primers and probes designed in this invention do not cross-react with other non-target pathogens commonly found in aquaculture environments, and can accurately identify three target pathogens: Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonidae. The detection method has extremely high specificity.

[0030] Example 4: Sensitivity and Standard Curve Verification To evaluate the sensitivity advantage of the multiplex qPCR detection method established in this invention, this embodiment uses serially diluted recombinant plasmids as standard templates to compare the detection limits of triplex qPCR and conventional PCR for Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonidae.

[0031] 1. Preparation of Standards Specific gene sequences of three pathogenic bacteria—Edwardsella tarda, Streptococcus dolphinus, and Aeromonas salmonicida—were ligated into the pUC57 vector to construct three recombinant plasmid standards, named sequentially: Edwardsiella tarda standard plasmid, Streptococcus dolphinus standard plasmid, and Aeromonas salmonicida standard plasmid. Plasmid construction was commissioned to Shanghai Bailig Biotechnology Co., Ltd.

[0032] The concentration was determined using a UV spectrophotometer, and the copy number of each plasmid was calculated based on its length and concentration. The calculation formula is as follows: (copies / μL) = [plasmid concentration (ng / L) × 10] -9 ×6.02×10 23 ] / (number of bases × 660) The copy number calculation results of plasmids are shown in Table 1: Table 1. Copy number of plasmids

[0033] Each standard plasmid was diluted to 1×10⁻⁶ using TE buffer. 9 copies / μL. Take equal volumes of three 1×10⁻⁶ copies / μL. 9 Mix the standard plasmid solution (copies / μL) and 7 times the volume of TE buffer thoroughly to obtain a mixed plasmid stock solution (each pathogen plasmid concentration is 1×10⁻⁶). 8 (copies / μL).

[0034] The mixed plasmid stock solution was serially diluted 10-fold with TE buffer to obtain concentrations of 1 × 10⁻⁶. 7 1×10 6 1×10 5 1 × 10 4 1×10 3 1 × 10 2 1 × 10 1 and 1 × 10 0 A series of standard templates in copies / μL (of which 1×10 0 (Copies / μL, i.e., 1 copy / μL). TE buffer was also set up as a negative control (0 copies / μL). These standards were used for subsequent sensitivity experiments and the establishment of a standard curve.

[0035] 2. Triple qPCR detection Using mixed standards at various concentration gradients as templates, amplification was performed according to the optimized multiplex qPCR reaction system and procedure determined in Example 2. After the reaction, the Ct values ​​for each channel were recorded. A standard curve was plotted with copy number logarithm on the x-axis and Ct values ​​on the y-axis, and the limit of detection was determined. The standard curve is shown below. Figure 2 As shown, in 10 1~6 Within the gradient concentration range of copies / L, the standard curves for Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonicida all showed good linearity. The obtained linear equations for copy number (x) and Ct value (y) and correlation coefficient R were also obtained. 2 for: Edwardsiella tarda: y = -3.421x + 39.95, R 2 =0.999; Streptococcus dolphinii: y = -3.501x + 39.84, R 2 =0.999; Kills Aeromonas salmonii: y = -3.403x + 39.79, R 2 =1.00. The fluorescence quantitative PCR amplification curve is as follows: Figure 3 As shown, Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonicidae can be detected normally at a concentration gradient of 1 copies / μL. Therefore, the detection rate of Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonicidae in this detection method can reach 1 copy / μL.

[0036] 3. Routine PCR testing Using single standards at various concentration gradients as templates, conventional PCR amplification was performed using pathogen-specific upstream and downstream primers (without probes). The PCR reaction system (50 μL) consisted of: 25 μL of 2X Accurate Taq Master Mix (dye plus), 2 μL each of 10 μM upstream and downstream primers, 2 μL of template, and ddH.O to a final volume of 50 μL. The PCR program was as follows: 94°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 15 s, for a total of 35 cycles; and a final extension at 72°C for 2 min. The amplified products were subjected to 2% agarose gel electrophoresis, and the target bands were observed using a gel imaging system.

[0037] 4. Sensitivity Comparison Compare the amplification curve of the FAM detection channel with the electrophoresis image of conventional PCR detection of Edwardsiella tarda ( ). Figure 4 -a), compare the amplification curve of the VIC detection channel with the electrophoresis image of the conventional PCR detection of Streptococcus dolphinus ( Figure 4 -b), compare the amplification curve of the ROX detection channel with the electrophoresis image of the conventional PCR detection of Aeromonas salmonidae ( Figure 4 -c). The comparison shows that the triple qPCR detection method established in this invention achieves a detection sensitivity of up to 1 copy / μL for *Edwardsiella tarda*, *Streptococcus dolphinus*, and *Aeromonas salmonicida* in three independent fluorescence detection channels, significantly higher than the conventional PCR methods for each pathogen (10). 3 (copies / μL), increasing sensitivity by up to 1000 times.

[0038] Example 5 Repeatability Test The recombinant plasmid standards for Edwardsiella tarda, Streptococcus dolphin, and Aeromonas salmonicida constructed in Example 4 were diluted with TE buffer to a concentration of 1.00 × 10⁻⁶. 6 copies / μL (high concentration), 1.00×10 4 copies / μL (medium concentration) and 1.00 × 10 2 Three levels of copies / μL (low concentration) were used. Deionized water was used as a template as a negative control.

[0039] Intra-batch replication: In the same batch of experiments, the plasmid standards at each concentration were tested three times, and amplification was performed according to the multiplex qPCR reaction system and procedure established in Example 2. The Ct values ​​of each test were recorded, and the mean, standard deviation (SD), and coefficient of variation (CV%) of Ct for each concentration level were calculated.

[0040] Inter-batch replication assays: Using different batches of qPCR premix at different times, the plasmid standards at each concentration were tested three times (three replicates per test), and amplification was performed according to the multiplex qPCR reaction system and procedure established in Example 2. The Ct values ​​of each test were recorded, and the mean, standard deviation (SD), and coefficient of variation (CV%) of Ct were calculated for each concentration level.

[0041] The results are shown in Table 2. The coefficient of variation for within-group repeated experiments ranged from 0.26% to 1.03%, and the coefficient of variation for between-group repeated experiments ranged from 0.36% to 1.18%, with all coefficients of variation less than 2.0%. The repeated experiment results indicate that the detection method established in this invention has good repeatability and stability.

[0042] Table 2 Repeatability Test Results

[0043] Example 6: Simulated Clinical Sample Testing Liver tissue from healthy fish that had previously been tested negative for Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonidae was obtained and homogenized under aseptic conditions.

[0044] Overnight cultures of Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonidae were serially diluted 10-fold to prepare concentrations of 1.0 × 10⁻⁶. 8 CFU / mL, 1.0 × 10 6 CFU / mL and 1.0×10 4 Three target bacterial dilutions at CFU / mL.

[0045] Following a volume ratio of fish liver homogenate to bacterial suspension of 9:1, the three concentrations of bacterial suspension mentioned above were added to the homogenate, and the mixture was thoroughly vortexed to prepare a simulated clinical infection sample. At this point, the final concentrations of the three target bacteria in the simulated sample were 1.0 × 10⁻⁶. 7 CFU / mL, 1.0 × 10 5 CFU / mL and 1.0 × 10 3 CFU / mL. Untreated fish liver homogenate was also used as a negative control. Additionally, pure cultures of *Edwards tarda*, *Streptococcus dolphinus*, and *Aeromonas salmonicida* at 1.0 × 10⁻⁶ CFU / mL were prepared. 7 CFU / mL, 1.0×10 5 CFU / mL and 1.0×10 3 Three concentration gradients of CFU / mL were used as positive controls.

[0046] After extracting DNA from each sample, the samples were tested according to the method established in Example 2. The results are shown in Table 3. The results show that the blank negative control had no specific detection signal; the added pathogens were accurately detected in simulated infection samples of all three target bacteria at various concentrations, and the consistency rate of the sample detection results was comparable to that of the pure cultures of each pathogen; the fish liver tissue matrix did not significantly interfere with the detection results. The results indicate that the detection primers and detection method of the present invention can effectively detect target pathogens in clinically suspected infection samples. Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonidae can be accurately detected in fish liver tissue matrix. The detection results are highly consistent with the pure cultures, and the tissue matrix does not significantly interfere with the detection, showing good prospects for practical application.

[0047] .

Claims

1. A multiplex qPCR primer set for detecting core pathogenic bacteria in aquaculture, characterized in that: The multiplex qPCR primer set includes primer combination A for detecting Edwardsiella tarda, primer combination B for detecting Streptococcus dolphinus, and primer combination C for detecting Aeromonas salmonicidae; wherein, The primer combination A for detecting Edwardsiella tarda includes: Upstream primer: 5'-GGGGTAAGTTCGACGACAAC-3' (SEQ ID NO.1) Downstream primer: 5'-GCGGATCACCAGTTCCAAC-3' (SEQ ID NO.2) Probe: 5'-CCTACAAGGTTTCCGGTGGTCTGC-3' (SEQ ID NO.3), the 5' end of the probe is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the fluorescent quencher group BHQ1; The primer combination B for detecting Streptococcus dolphinus includes: Upstream primer: 5'-TCCAGCTAACAACGGTCAA-3' (SEQ ID NO.4) Downstream primer: 5'-CTTCCTCTTTCGCTTTACGG-3' (SEQ ID NO.5) Probe: 5'-AGAGTACCAGCTCCAACCATAACCGC-3' (SEQ ID NO.6), the 5' end of the probe is labeled with a fluorescent reporter group VIC, and the 3' end is labeled with a fluorescent quencher group BHQ1; The primer combination C for detecting Aeromonas salmonicida includes: Upstream primer: 5'-ACTGTCTGTTACCCTGCCAAG-3' (SEQ ID NO.7) Downstream primer: 5'-CAGCAGTGATAGAAGCACCAAC-3' (SEQ ID NO.8) Probe: 5'-CAGCAGCAACATCAGCAGGCTTCA-3' (SEQ ID NO.9), the 5' end of the probe is labeled with the fluorescent reporter group ROX, and the 3' end is labeled with the fluorescent quencher group BHQ2.

2. The use of the multiplex qPCR primer set according to claim 1 in the preparation of a kit for the simultaneous detection of Edwardsiella tarda, Streptococcus dolphinus, and Aeromonas salmonicida.

3. A kit for detecting core pathogenic bacteria in aquaculture, the kit being capable of simultaneously detecting Edwardsiella tarda, Streptococcus dolphinii, and Aeromonas salmonicida, characterized in that: The kit includes the multiplex qPCR primer set as described in claim 1.

4. The kit according to claim 3, characterized in that, The kit also includes: (a) qPCR reaction system premix: The premix contains DNA polymerase, dNTPs and PCR buffer; (b) Positive control: The positive control is a recombinant plasmid containing target gene fragments of Edwardsiella tarda, Streptococcus dolphinus and Aeromonas salmonicidae; (c) Negative control: The negative control is nuclease-free water.

5. A method for detecting core pathogenic bacteria in aquaculture using the multiplex qPCR primer set described in claim 1, comprising the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted DNA as a template, multiplex qPCR amplification was performed using a set of multiplex qPCR primers; (3) Collect fluorescence signals, and determine the results based on the amplification curve and Ct value as follows: a. The positive / negative status of the FAM channel: Positive: When a typical amplification curve appears in the FAM channel amplification result of the test sample and Ct ≤ 35, it is determined that the test sample contains Edwardsiella tarda; Negative: When there is no amplification curve in the FAM channel amplification result of the test sample, or although there is an amplification curve in the FAM channel amplification result of the test sample but Ct ≥ 38, it is determined that the test sample does not contain Edwardsiella tarda; Suspicious: When an amplification curve appears in the FAM channel amplification result of the test sample and 35 < Ct < 38, it is determined that the test sample is a suspicious sample and needs to be retested after re-extracting DNA; b. Positive and negative situations of the VIC channel: Positive: When a typical amplification curve appears in the VIC channel amplification result of the test sample and Ct ≤ 35, it is determined that the test sample contains Streptococcus iniae; Negative: When there is no amplification curve in the VIC channel amplification result of the test sample, or although there is an amplification curve in the VIC channel amplification result of the test sample but Ct ≥ 38, it is determined that the test sample does not contain Streptococcus iniae; Suspicious: When an amplification curve appears in the VIC channel amplification result of the test sample and 35 < Ct < 38, it is determined that the test sample is a suspicious sample and needs to be retested after re-extracting DNA. c. Positive and negative situations of the ROX channel: Positive: When a typical amplification curve appears in the ROX channel amplification result of the test sample and Ct ≤ 35, it is determined that the test sample contains Aeromonas salmonicida; Negative: When there is no amplification curve in the ROX channel amplification result of the test sample, or although there is an amplification curve in the ROX channel amplification result of the test sample but Ct ≥ 38, it is determined that the test sample does not contain Aeromonas salmonicida; Suspicious: When an amplification curve appears in the ROX channel amplification result of the test sample and 35 < Ct < 38, it is determined that the test sample is a suspicious sample and needs to be retested after re-extracting DNA.

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