Fluorescent PCR (Polymerase Chain Reaction) primer, probe and kit for detecting aeromonas hydrophila of silver carp and bighead carp as well as detection method and application thereof

Abstract

The invention relates to the technical field of biological detection, and discloses a primer, a probe and a kit for detecting aeromonas hydrophila based on fluorescent PCR (Polymerase Chain Reaction) and application of the primer, the probe and the kit. A primer and fluorescent probe combination is obtained by designing and screening a specific gene conserved region of aeromonas hydrophila. The specific amplification of the aeromonas hydrophila gene based on the fluorescent PCR technology can be successfully realized. According to the method, the detection accuracy and sensitivity are improved, the aeromonas hydrophila can be detected in the early stage of bacterial septicemia of silver carps and bighead carps, prevention and control of bacterial septicemia of silver carps and bighead carps are facilitated, and the basic requirements of field detection of farms can be met.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically a fluorescent PCR primer, probe, kit, detection method and application for detecting Aeromonas hydrophila in silver carp and bighead carp. Background Technology

[0002] Silver carp and bighead carp are filter-feeding fish with important ecological and economic value in my country's freshwater aquaculture. Hypophthalmichthys molitrix ) and bighead carp ( Aristichthys nobilis Aeromonas hydrophila effectively regulates eutrophication in aquaculture waters by feeding on plankton, making it a key species in pond polyculture. It grows rapidly and is highly adaptable, accounting for a significant share of my country's major freshwater fish production. In recent years, with increased stocking densities, Aeromonas hydrophila (…) has become a major species in polyculture. Aeromonas hydrophila Frequent outbreaks of bacterial septicemia and enteritis caused by Aeromonas hydrophila (AGH) result in infected fish exhibiting external bleeding, swollen internal organs, and ascites, leading to high mortality rates and significant economic losses. Furthermore, Aeromonas hydrophila is a typical zoonotic pathogen, and its public health implications cannot be ignored. This bacterium not only harms aquatic animals but can also infect humans through contact with contaminated water sources or consumption of undercooked infected aquatic products, making it an important foodborne and opportunistic pathogen. Human infection can cause skin wound infections, gastroenteritis, and diarrhea; in severe cases, it can even lead to necrotizing fasciitis or septicemia, posing a particularly significant threat to immunocompromised populations. Therefore, establishing rapid and accurate detection methods for Aeromonas hydrophila is not only necessary for ensuring the healthy development of the aquaculture industry but also a crucial step in controlling food safety risks and maintaining public health security.

[0003] Currently, the detection of Aeromonas hydrophila mainly relies on bacterial isolation, culture, and biochemical identification, which is cumbersome and time-consuming. In addition, the ELISA detection method established by Ren Yan et al. (Ren Yan, Pan Zihao, Lu Chengping, Yao Huochun, Wu Shuqin. Detection of pathogenic Aeromonas hydrophila by Dot-ELISA method [J]. Journal of Animal Husbandry and Veterinary Medicine, 2011, 42 (10): 1409-1415.) is relatively fast, but its sensitivity is limited and it is easily affected by cross-reaction. The conventional PCR and isothermal amplification techniques (such as RPA and LAMP) disclosed in patents CN103305613B, CN117089638A, CN111793702A, CN110734994B, and CN119506444A have improved the detection sensitivity, but there are still problems such as complex primer design, unstable amplification specificity, or easy false positives, which are difficult to meet the needs of rapid and accurate diagnosis in fish farms.

[0004] Therefore, it is necessary to develop a simpler method for detecting Aeromonas hydrophila. By designing specific primers and probes, a highly sensitive, specific, and rapid quantitative detection method for Aeromonas hydrophila in silver carp and bighead carp can be achieved, providing a reliable technical means for early diagnosis and control in aquaculture. Summary of the Invention

[0005] To effectively overcome the shortcomings and limitations of the above-mentioned technologies, this invention establishes a detection method based on fluorescence PCR (Quantitative PCR, qPCR), wherein the primers used are: upstream primer F selected from any one of SEQ ID NO.1-SEQ ID NO.5; and downstream primer R selected from any one of SEQ ID NO.6-SEQ ID NO.10.

[0006] Furthermore, it also includes probe P, which is selected from any one of SEQ ID NO.11-SEQ ID NO.15.

[0007] Furthermore, the primer set gene sequence is arranged from left to right, with the left side being the start end (5') and the right side being the extension end (3').

[0008] Furthermore, the gene sequence of the probe is arranged from left to right, with the left side being the start end 5' and the right side being the extension end 3'. The start end 5' is labeled with a fluorescent gene, and the extension end 3' is labeled with a quenching gene.

[0009] Furthermore, the fluorescent group is FAM, and the quenching group is BHQ. BHQ is a quencher produced by LGC Corporation, and there are BHQ types 1-3. This invention can use any of the quenchers of type 1-3.

[0010] The present invention also provides a real-time fluorescent kit for detecting Aeromonas hydrophila in silver carp and bighead carp, comprising the above-mentioned primer set and probe.

[0011] Furthermore, the kit also includes plasmid standards. These plasmid standards are constructed using the pcDNA3.1 plasmid as a vector, into which the Aeromonas hydrophila-specific gene gyrA is inserted via genetic engineering methods; the nucleotide sequence of the specific gene gyrA is shown in SEQ ID NO.16.

[0012] The detection method using the above probe, primer set, and kit includes the following steps: (1) taking the sample to be tested and extracting DNA; (2) Using the extracted DNA as a template, perform real-time PCR using the upstream primer F, downstream primer R, and fluorescent probe P; (3) If a fluorescent signal is present, the sample is positive; otherwise, it is negative.

[0013] Furthermore, in step (2), the reaction system for real-time PCR is as follows: 10 μL of 2×Taq PCR Master Mix, 0.5 μL each of fluorescent probe, upstream primer and downstream primer, 1 μL of DNA template to be tested or plasmid standard, and 20 μL of sterile ddH2O. The reaction program was as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 60℃ annealing / extension for 34s; 40 cycles.

[0014] Furthermore, the present invention also provides the application of the kit, (1) qualitative detection and / or analysis of Aeromonas hydrophila in silver carp and bighead carp; (2) Monitor the bacterial load in aquaculture water and feed to provide early warning of the risk of bacterial septicemia outbreaks in silver carp and bighead carp; (3) Quantitative analysis of bacterial load in diseased fish to aid in disease grading, disease course assessment and prognosis; (4) Evaluate the efficacy of disinfectants and antibacterial drugs to guide precise drug use and reduce antibiotic abuse; (5) Assess the safety of the micro-ecology of the aquaculture environment to help optimize water quality control and aquaculture management models; (6) Compliance screening of pathogens for aquatic products before they are put on the market to ensure food safety and market circulation; (7) Epidemiological investigation of Aeromonas hydrophila to trace transmission routes and regional epidemic patterns; (8) Quantitative detection of virulence genes in bacterial strains to assess pathogenicity and outbreak risk; (9) Research on disease-resistant breeding of silver carp and bighead carp to assist in screening high-quality germplasm resources resistant to Aeromonas hydrophila.

[0015] In summary, the beneficial effects obtained by adopting the above technical solution are as follows: 1. High detection efficiency and safe and convenient operation: Based on the probe-based real-time fluorescence quantitative PCR technology, this invention eliminates the need for electrophoresis detection steps and can complete the entire detection process within 1–2 hours, significantly shortening the detection cycle. At the same time, it effectively avoids the risk of amplification product contamination caused by the electrophoresis step, making it suitable for rapid on-site screening as well as meeting the high-throughput detection needs of large batches of samples.

[0016] 2. High detection specificity and reliable results: The primers and probes used in this invention are designed and screened for specific conserved regions of Aeromonas hydrophila. They have no cross-reaction with 7 common aquatic pathogens such as Flavobacterium psychrophilum and Aeromonas vernix, which can effectively avoid false positive results and significantly improve the accuracy and reliability of the detection results.

[0017] 3. High detection sensitivity and good stability: This invention can achieve accurate detection of low-copy target molecules, with a detection sensitivity of up to the picogram level, which is far superior to traditional PCR and LAMP detection methods. It can stably detect Aeromonas hydrophila in early infection or low-load samples, solving the problem of low detection rate of low-concentration pathogens in traditional methods.

[0018] 4. It can achieve precise quantification and has high industrial application value: This invention can accurately quantify the load of Aeromonas hydrophila in samples through a standard curve. It can not only complete the qualitative detection of pathogens, but also provide accurate data support for the assessment of the disease process of Aeromonas hydrophila infection in silver carp and bighead carp, early warning of outbreak risk, formulation of prevention and control strategies and evaluation of drug efficacy. It has outstanding practical application and industrial promotion value. Attached Figure Description

[0019] Figure 1 To screen the fluorescence signal curves and electrophoresis images of five primer-probe combinations, among which, Figure 1 A represents the primer and probe selection for gyrA. Figure 1 C is the electrophoresis diagram of gyrA gene screening. 1 represents the combination of upstream primer F1, downstream primer R1 and probe P1; 2 represents the combination of upstream primer F2, downstream primer R2 and probe P2; 3 represents the combination of upstream primer F3, downstream primer R3 and probe P3; 4 represents the combination of upstream primer F4, downstream primer R4 and probe P4; and 5 represents the combination of upstream primer F5, downstream primer R5 and probe P5.

[0020] Figure 2 The present invention provides a standard curve and sensitivity test results for the primers and probes used in the detection of Aeromonas hydrophila using real-time PCR. Figure 2 A is the standard curve. Figure 2 B shows the detection results of plasmids at different concentrations (1, 2, 3, and 4 represent 1×10⁻⁶ plasmids, respectively). 9 1×10 8 1×10 7 and 1×10 6 (copies / μL).

[0021] Figure 3 The lowest detection limit (1×10⁻⁶) for Aeromonas hydrophila was determined in Example 3. -2 Amplification results (copies / μL).

[0022] Figure 4The results of the specificity test of the primers and probes of this invention for the detection of Aeromonas hydrophila are shown in Figure A, which is a fluorescence signal curve and Figure B is an electrophoresis diagram (M: Marker, 1: negative control ddH2O, 2: positive control standard plasmid, 3: Aeromonas hydrophila, 4: Flavobacterium psychrophilum, 5: Aeromonas versicolor, 6: Edwardsiella tarda, 7: Shewanella putrefactive, 8: Aeromonas temperate, 9: Staphylococcus aureus, 10: Escherichia coli).

[0023] Figure 5 The gyrA gene (MZ494456.1) of Aeromonas hydrophila and Escherichia coli were identified. Escherichia coli gyrA (MN148181.1), Flavobacterium columnare Flavobacterium columnare gyrA (PV820829.1), psychrophilic flavipes gyrA (AB158127.1), and Nocardia africana. Nocardia transvalensis gyrA (KM194606.1), Shigella Shigella dysenteriae Sequence comparison of the gyrA gene (AY648051.1). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0025] Example 1: 1. Obtaining positive plasmids of Aeromonas hydrophila: Aeromonas hydrophila genes were searched by NCBI, and the Aeromonas hydrophila-specific gene gyrA was selected as the target gene and constructed into the vector pcDNA3.1. The sequence of gyrA is shown in SEQ ID NO.16.

[0026] 2. Five primer pairs and five probes were designed targeting the specific target gene gyrA of Aeromonas hydrophila, the main pathogen of bacterial septicemia in silver carp and bighead carp. The primer sequences are shown in SEQ ID NO.1-15. The gene sequence of the probes is from left to right, with the left side being the start end 5' and the right side being the extension end 3'. The start end 5' is labeled with the fluorescent group FAM, the extension end 3' is labeled with the quencher group BHQ, the quencher group for P1-P3 is BHQ-1, and the quencher group for P4-P5 is BHQ2-3.

[0027] 3. Detection of Aeromonas hydrophila was performed using quantitative real-time PCR. The sample was silver carp blood. A plasmid containing the Aeromonas hydrophila gyrA gene sequence was synthesized as a template. The reaction system is as follows: 10 μL of 2×Taq PCR Master Mix, 0.5 μL each of fluorescent probe P (10 μM), upstream primer F (10 μM), and downstream primer R (10 μM), and gyrA plasmid (1×10⁻⁶). 7 1 μL (copies / μL) was added and brought to a final volume of 20 μL with sterile ddH2O.

[0028] The reaction program for real-time PCR is as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 60℃ annealing / extension for 34s; 40 cycles.

[0029] Record the data and perform gel electrophoresis; the results are as follows: Figure 1 As shown, the upstream and downstream primers, as well as the probe, can specifically amplify the target gene. The most suitable primer combination is determined to be F4R4 based on the intensity of the fluorescence signal curve.

[0030] Example 2: Using the probe P4, primer set F4 and R4, detection system, and procedure of this invention, quantitative analysis of *Aeromonas hydrophila* can also be performed. The sample tested in this example is silver carp blood. The probe, primer set, detection system, and procedure are consistent with Example 1: A standard curve was established. The plasmid concentration containing the *Aeromonas hydrophila* gyrA gene was determined using an ultra-micro spectrophotometer. The corresponding copy number was calculated using the following formula: copies / μL = (ng / μL × 10⁻⁶) -9 ) × (6.022 × 10 23 ) / (DNA length × 660). Dilute the plasmid stock solution 10-fold with sterile ddH2O (1 × 10⁻⁶). 8 ~1×10 4 The template plasmid standard (copies / μL) was used for detection using the real-time PCR system and reaction procedure established in Example 1. Fluorescence data were recorded and a standard curve was plotted.

[0031] The equation of the standard curve is: y = -32.8661x + 36.222, R ∈ R. 2 = 0.9975. Its Ct value shows a good linear relationship with the concentration of the standard plasmid, as shown in the results. Figure 2 As shown, the plasmid concentrations differ by a factor of 10, and the Ct values ​​differ by approximately 3.1, which is consistent with the theoretical values.

[0032] Example 3: In this example, the sensitivity of the primers was detected, specifically using the P4 probe and primer set F4 and R4.

[0033] The gyrA plasmid of Aeromonas hydrophila shown in Example 1 was diluted to 1×10⁻⁶. -1 copies / μL, 1×10 -2 After amplification at a concentration of copies / μL, the lowest concentration with a detection rate of over 95% is selected as the detection line concentration.

[0034] The results are as follows Figure 3 As shown, the detection limit of the primers for Aeromonas hydrophila gyrA is 1 × 10⁻⁶. -2 copies / μL.

[0035] Example 4: This example provides a kit for detecting Aeromonas hydrophila based on real-time quantitative PCR, which includes the primer and probe combination P4, F4, and R4, the reaction system, and the reaction procedure provided in Example 1. This experimental example verifies the application and evaluates the specificity of the detection kit. The specific operation is as follows: 1. Prepare DNA templates: Select eight common pathogenic bacteria, including Aeromonas hydrophila, Aeromonas vera, Flavobacterium psychrophilum, Edwardsiella tarda, Shewanella putrefactive, Aeromonas sobria, Staphylococcus aureus, and Escherichia coli. Extract DNA using a bacterial genomic DNA extraction kit (purchased from the manufacturer), with a concentration of 1×10⁻⁶ for each sample. 3 The specificity of the kit was evaluated by using pg / μL of pathogen DNA as a template, with gyrA plasmid standard as a positive control and ddH2O as a negative control.

[0036] 2. Perform real-time PCR amplification according to the primer and probe combination, amplification reagents, reaction system and reaction procedure provided in Example 1.

[0037] 3. Results are as follows Figure 4 As shown, only the positive plasmid standard and *Aeromonas hydrophila* exhibited fluorescence amplification curves; *Flavobacterium psychrophilum*, *Aeromonas verrucosa*, *Edwards tarda*, *Shewanella putrefactiveis*, *Aeromonas temperate*, *Staphylococcus aureus*, *Escherichia coli*, and the negative control showed no fluorescence amplification curves. The gene sequence alignment table for each bacterium is shown below. Figure 5 This demonstrates that the primer-probe combination contained in this invention has high specificity and can specifically detect Aeromonas hydrophila.

[0038] Therefore, it can be seen from the above embodiments that the primer set, probe, and detection method of the present invention are used in the following applications: (1) Qualitative and quantitative detection and / or analysis of Aeromonas hydrophila in silver carp and bighead carp; (2) Monitor the bacterial load in aquaculture water and feed to provide early warning of the risk of bacterial septicemia outbreaks in silver carp and bighead carp; (3) Quantitative analysis of bacterial load in diseased fish to aid in disease grading, disease course assessment and prognosis; (4) Evaluate the efficacy of disinfectants and antibacterial drugs to guide precise drug use and reduce antibiotic abuse; (5) Assess the safety of the micro-ecology of the aquaculture environment to help optimize water quality control and aquaculture management models; (6) Compliance screening of pathogens for aquatic products before they are put on the market to ensure food safety and market circulation; (7) Epidemiological investigation of Aeromonas hydrophila to trace transmission routes and regional epidemic patterns; (8) Quantitative detection of virulence genes in bacterial strains to assess pathogenicity and outbreak risk; (9) Research on disease-resistant breeding of silver carp and bighead carp to assist in screening high-quality germplasm resources resistant to Aeromonas hydrophila.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A fluorescent quantitative PCR primer for detecting Aeromonas hydrophila in silver carp and bighead carp, characterized in that, The upstream primer F is selected from any one of SEQ ID NO.1-SEQ ID NO.5; The downstream primer R is selected from any one of SEQ ID NO.6-SEQ ID NO.10; The primers have the following gene sequence order from left to right: the left side is the start end (5') and the right side is the extension end (3').

2. A fluorescent quantitative probe for detecting Aeromonas hydrophila in silver carp and bighead carp, characterized in that, The probe P is selected from any one of SEQ ID NO. 11-SEQ ID NO. 15; The gene sequence of the probe is arranged from left to right, with the left side being the start end 5' and the right side being the extension end 3'. The start end 5' is labeled with a fluorescent group, and the extension end 3' is labeled with a quenching group.

3. The probe according to claim 2, characterized in that, The fluorescent group is FAM, and the quenching group is BHQ.

4. A real-time fluorescent reagent kit for detecting Aeromonas hydrophila in silver carp and bighead carp, characterized in that, Includes the primers and probes described in claims 1 and 2.

5. The reagent kit according to claim 4, characterized in that, It also includes plasmid standards.

6. The reagent kit according to claim 5, characterized in that, The plasmid standard was constructed by inserting the Aeromonas hydrophila-specific gene gyrA into the pcDNA3.1 plasmid using genetic engineering methods; the nucleotide sequence of the specific gene gyrA is shown in SEQ ID NO.

16.

7. A method for quantitative fluorescence detection of Aeromonas hydrophila in silver carp and bighead carp, characterized in that, The steps include: (1) taking the sample to be tested and extracting DNA; (2) Using the extracted DNA as a template, fluorescent PCR detection was performed using the upstream primer F and downstream primer R described in claim 1, and the fluorescent probe P described in claim 2; (3) If a fluorescent signal is present, the sample is positive; otherwise, it is negative.

8. The method according to claim 7, characterized in that, In step (2), the reaction system for fluorescent PCR is as follows: 10 μL of 2×Taq PCR Master Mix, 0.5 μL each of fluorescent probe, upstream primer and downstream primer, 1 μL of DNA template to be tested or plasmid standard as described in claim 5, and 20 μL to be supplemented with sterile ddH2O. The reaction program was as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 60℃ annealing / extension for 34s; 40 cycles.

9. The application of the reagent kit according to claim 4, characterized in that, The kit can be used for the following applications: (1) Qualitative detection and / or analysis of Aeromonas hydrophila in silver carp and bighead carp; (2) Monitor the bacterial load in aquaculture water and feed to provide early warning of the risk of bacterial septicemia outbreaks in silver carp and bighead carp; (3) Quantitative analysis of bacterial load in diseased fish to aid in disease grading, disease course assessment and prognosis; (4) Evaluate the efficacy of disinfectants and antibacterial drugs to guide precise drug use and reduce antibiotic abuse; (5) Assess the safety of the micro-ecology of the aquaculture environment to help optimize water quality control and aquaculture management models; (6) Compliance screening of pathogens for aquatic products before they are put on the market to ensure food safety and market circulation; (7) Epidemiological investigation of Aeromonas hydrophila to trace transmission routes and regional epidemic patterns; (8) Quantitative detection of virulence genes in bacterial strains to assess pathogenicity and outbreak risk; (9) Research on disease-resistant breeding of silver carp and bighead carp to assist in screening high-quality germplasm resources resistant to Aeromonas hydrophila.

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