Primer probe and kit for detecting pathogenic shewanella putrefaciens and application thereof

Abstract

The present application relates to the technical field of biological detection, and particularly relates to a primer probe, a kit and an application for detecting pathogenic shewanella putrefaciens. The present application takes the gyrB gene of shewanella putrefaciens as a target, and a specific primer and a fluorescent probe combination are obtained through screening, so that the rapid detection of shewanella putrefaciens by fluorescence quantitative PCR can be successfully realized. The method has the advantages of high sensitivity, strong specificity, good repeatability and the like, has no cross reaction with other common aquatic pathogenic bacteria, can realize the accurate screening of pathogens in the early stage of bacterial diseases of silver carp and bighead carp, and provides a reliable technical means for the early warning and prevention and control of the epidemic caused by shewanella putrefaciens in aquaculture production.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a primer probe and kit for detecting pathogenic Shewanella putrefactive bacteria, and its application. Background Technology

[0002] Shewanella putrefaction Shewanella putrefaciens It is a potentially dangerous facultative anaerobic Gram-negative bacillus belonging to the Vibrioceae family. Vibrionaceae Shewanella is a genus of bacteria that is widely distributed in freshwater, sewage, and marine environments. It is one of the typical spoilage bacteria in aquatic animals and protein-rich foods. In the field of food safety, Shewanella putrefaction has long been recognized as one of the dominant spoilage bacteria in low-temperature stored aquatic products. It can maintain its metabolic activity in cold storage environments below 4°C, causing fish, shrimp, shellfish and other aquatic products to spoil and deteriorate during storage and transportation, resulting in serious economic losses.

[0003] In recent years, increasing research evidence suggests that the pathogenicity of Shewanella putrefaction is no longer limited to causing spoilage of aquatic products. Its harmfulness as a primary pathogen in farmed aquatic animals is gradually attracting attention, and cases of infection with this bacterium in various economically important fish species have been reported both domestically and internationally, including crucian carp. Carassius auratus Infection with this bacterium resulted in obvious symptoms of hemorrhagic septicemia; *Shewanella putrefactiveis* was also detected in bacterial septicemia outbreaks in silver carp during the early spring after hibernation; in addition, largemouth bass... Micropterus salmoides ,loach Misgurnus anguillicaudatus Giant Salamander Andrias davidianus and rainbow trout Oncorhynchus mykiss The pathogenicity of this bacterium has also been reported in other species. More importantly, *Shewanella putrefaction* is an opportunistic pathogen that can be transmitted between humans, animals, and fish. Immunocompromised individuals can become infected through contact with contaminated water sources via wounds or by consuming undercooked aquatic products carrying the bacterium, leading to severe clinical symptoms such as soft tissue necrosis, bacteremia, and even endocarditis. Therefore, developing a method for early, rapid, and accurate detection of *Shewanella putrefaction* in silver carp and bighead carp and their aquaculture environment is not only an urgent need for disease control in aquaculture but also crucial for preventing aquatic product quality and safety risks and protecting public health.

[0004] Currently, the detection of Shewanella putrefaction mainly relies on bacterial isolation and culture and biochemical identification, which is cumbersome and time-consuming. Existing studies have shown that by the time the identification process is completed using traditional isolation and identification methods, more than half of the farmed fish have already died. Although some researchers have established routine PCR detection methods, such as Zhao Yanhua et al. who developed PCR primer pairs and detection kits for pathogenic Shewanella putrefaction in crucian carp, the following shortcomings still exist: First, routine PCR requires gel electrophoresis to determine the results, which is cumbersome and carries the risk of contamination; second, it lacks quantitative detection capabilities and cannot accurately determine the pathogen load; and third, the primer design is mainly based on the 16S rRNA gene, and the specificity needs to be further improved.

[0005] To address the aforementioned problems, this invention introduces fluorescently labeled specific probes into the reaction system using quantitative real-time PCR (qPCR) technology, enabling real-time monitoring and absolute quantitative analysis of the amplification process. Compared to conventional PCR, qPCR offers three major advantages: First, closed-tube detection eliminates the need for electrophoresis and opening the tube, significantly reducing the risk of laboratory contamination; second, the use of specific probes provides stronger specificity than the dual-recognition mechanism of ordinary primers, effectively avoiding false positives caused by non-specific amplification; and third, it offers a wide quantitative range and high sensitivity, capable of detecting 1-10... 1 Stable detection of trace amounts of pathogenic nucleic acid at the copy / μL level has been achieved. To date, no patent reports have been found regarding the detection of *Shewanella putrefactiveis* from silver carp and bighead carp using the TaqMan probe-based real-time PCR method.

[0006] Based on the above analysis, this invention uses the gyrB gene of Shewanella putrefactive bacteria as a molecular target, designs and screens a set of specific primers and TaqMan fluorescent probes, and establishes a fluorescent quantitative PCR method suitable for rapid quantitative detection of Shewanella putrefactive bacteria in silver carp and bighead carp samples. It aims to provide a simple, reliable, and early warning pathogen detection technology solution for front-line aquaculture. Summary of the Invention

[0007] In view of this, the present invention provides a primer probe and kit for detecting pathogenic Shewanella putrefactive bacteria, aiming to solve the problem of rapid screening and early warning of bacterial pathogens in silver carp and bighead carp during aquaculture.

[0008] This invention provides a primer probe for detecting pathogenic Shewanella putrefactive bacteria, the primer probe being composed of any one of the primer probes (1)-(5): (1) The upstream primer sequence is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:6, and the fluorescent probe sequence is shown in SEQ ID NO:11; (2) The upstream primer sequence is shown in SEQ ID NO:2, the downstream primer sequence is shown in SEQ ID NO:7, and the fluorescent probe sequence is shown in SEQ ID NO:12; (3) The upstream primer sequence is shown in SEQ ID NO:3, the downstream primer sequence is shown in SEQ ID NO:8, and the fluorescent probe sequence is shown in SEQ ID NO:13; (4) The upstream primer sequence is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:9, and the fluorescent probe sequence is shown in SEQ ID NO:14; (5) The upstream primer sequence is shown in SEQ ID NO:5, the downstream primer sequence is shown in SEQ ID NO:10, and the fluorescent probe sequence is shown in SEQ ID NO:15.

[0009] Preferably, the probe sequence is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quencher group at the 3' end.

[0010] The present invention also provides the application of primer probes in the preparation of products for detecting pathogenic Shewanella putrefactive bacteria.

[0011] Preferably, the product is a reagent or kit.

[0012] Preferred application methods include: S1. Take the sample to be tested and extract DNA; S2. Using the extracted DNA as a template, perform real-time PCR using primer and probe combinations; S3. Result determination: If a fluorescent signal appears, the sample is positive, indicating the presence of Shewanella putrefactive bacteria in the sample.

[0013] Preferably, the reaction system for real-time PCR in S2 includes 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, and 7.5 μL of sterile ddH2O.

[0014] Preferably, the reaction program for real-time PCR is 95℃ pre-denaturation for 30s, 95℃ denaturation for 5s, 60℃ annealing / extension for 34s, for 40 cycles.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. Using the gyrB gene as the detection target, this gene is highly conserved within Shewanella putrefactive bacteria and shows significant interspecies differences, which is superior to traditional 16S rRNA targets; 2. The primer-probe combination showed no cross-reaction with seven common aquatic pathogens, including Aeromonas versicolor, Aeromonas hydrophila, Flavobacterium psychrophilum, Edwardsiella tarda, Aeromonas temperate, Staphylococcus aureus, and Escherichia coli. Only Shewanella putrefactiveis showed a specific amplification signal, demonstrating excellent species-level recognition ability. 3. Detection limit can reach 10. 1 The copy number / μL is far superior to traditional PCR methods and can meet the detection needs of trace pathogen nucleic acids in the early stage of infection. 4. The closed-tube detection strategy eliminates the need for electrophoresis and opening the tube throughout the entire process, significantly reducing the risk of contamination. The process from DNA extraction to result reporting for a single sample can be completed in about 1 hour, making it suitable for rapid screening applications in aquaculture sites or grassroots laboratories. 5. The degenerate base design introduced in the primers and probes enables them to adapt to the natural sequence variations of the gyrB gene in different Shewanella putrefactive strains, resulting in a broad strain recognition spectrum and applicability to the detection of Shewanella putrefactive isolates from different breeding regions in my country. Attached Figure Description

[0016] Figure 1 Fluorescence signal curves and electrophoresis diagrams were generated to screen five primer and probe combinations. A shows the primer screening diagram for gyrB, where 1 represents F1 and R1, 2 represents F2 and R2, 3 represents F3 and R3, 4 represents F4 and R4, and 5 represents F5 and R5. B shows the electrophoresis diagram of the primer screening for gyrB, where 1 represents F1 and R1, 2 represents F2 and R2, 3 represents F3 and R3, 4 represents F4 and R4, and 5 represents F5 and R5. C shows the probe screening diagram for gyrB, where 1 represents P1, 2 represents P2, 3 represents P3, and 4 represents P5. Figure 2 The graph shows the standard curve and sensitivity test results of real-time PCR for the detection of Shewanella putrefactive bacteria using primers and probes. In the graph, A is the standard curve and B is the detection results for different concentrations of plasmid. Figure 3 Amplification results for determining the limit of detection of Shewanella putrefactive bacteria; Figure 4 The graph shows the specificity test results of primers and probes for the detection of Shewanella putrefactive bacteria. In the graph, A is the fluorescence signal curve and B is the electrophoresis diagram. Figure 5This is a sequence comparison diagram of the gyrB gene NZ_CP066370.1 of Shewanella putrefactiveis with those of gyrBAY101807.1 of Aeromonas tempera, gyrB MW352997.1 of Flavobacterium columnare, gyrB EU259314.1 of Edwardsiella tarda, gyrB PV738889.1 of Pantothecinus pineapple, gyrB KY126152.1 of Acinetobacter baumannii, gyrBAY258264.1 of Pasteurella multocida, gyrB MW353002.1 of Flavobacterium kovalifolium, and gyrB AB034222.1 of Flavobacterium johnsonii. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps described in these embodiments do not limit the scope of this application.

[0018] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the scope of this application and its application or use.

[0019] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.

[0020] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] This invention provides a primer probe for detecting pathogenic Shewanella putrefactive bacteria, the primer probe being composed of any one of the primer probes (1)-(5): (1) The upstream primer sequence is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:6, and the fluorescent probe sequence is shown in SEQ ID NO:11; (2) The upstream primer sequence is shown in SEQ ID NO:2, the downstream primer sequence is shown in SEQ ID NO:7, and the fluorescent probe sequence is shown in SEQ ID NO:12; (3) The upstream primer sequence is shown in SEQ ID NO:3, the downstream primer sequence is shown in SEQ ID NO:8, and the fluorescent probe sequence is shown in SEQ ID NO:13; (4) The upstream primer sequence is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:9, and the fluorescent probe sequence is shown in SEQ ID NO:14; (5) The upstream primer sequence is shown in SEQ ID NO:5 and the downstream primer sequence is shown in SEQ ID NO:10.

[0023] In this invention, the upstream primer sequence is shown in SEQ ID NO:3, the downstream primer sequence is shown in SEQ ID NO:8, and the fluorescent probe sequence is shown in SEQ ID NO:13.

[0024] In the primer sequences described above, Y represents C / T degenerate bases, R represents A / G degenerate bases, M represents A / C degenerate bases, and K represents G / T degenerate bases. The design of degenerate bases enables the primer probes to efficiently identify synonymous mutation sites of the gyrB gene among different Shewanella putrefactive strains, significantly broadening the strain coverage of the detection method.

[0025] In this invention, the probe sequence is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quenching group at the 3' end.

[0026] The present invention also provides the application of primer probes in the preparation of products for detecting pathogenic Shewanella putrefactive bacteria.

[0027] In this invention, the product is a reagent or a kit.

[0028] In this invention, the kit includes a primer and probe set, a premixed solution for real-time PCR reaction, a positive plasmid standard, and a negative control.

[0029] In this invention, the premixed solution for quantitative real-time PCR reaction includes Taq DNA polymerase, dNTPs, ROX reference dye, and Mg. 2+ and PCR buffer; The positive plasmid standard is a pcDNA3.1 recombinant plasmid containing the target fragment of the gyrB gene of Shewanella putrefactive bacteria. It is constructed by inserting the gyrB gene containing the target gene into the pcDNA3.1 plasmid using genetic engineering methods. The target fragment sequence is shown in SEQ ID NO:16. The negative control was sterile ddH2O.

[0030] In this invention, the application method includes: S1. Take the sample to be tested and extract DNA; S2. Using the extracted DNA as a template, perform real-time PCR using primer and probe combinations; S3. If the sample to be tested shows a typical S-type fluorescence amplification curve and the Ct value is ≤35, it is judged as positive for Shewanella putrefactive bacteria nucleic acid detection; if there is no obvious fluorescence amplification curve or the Ct value is >35, it is judged as negative.

[0031] In this invention, the reaction system for real-time PCR in S2 includes 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, and 7.5 μL of sterile ddH2O.

[0032] In this invention, the reaction program for real-time PCR is 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing / extension for 34 s, for 40 cycles.

[0033] Example 1 This embodiment aims to screen the optimal primer pairs and specific probes suitable for detecting Shewanella putrefactive bacteria, a pathogenic bacterium found in silver carp and bighead carp.

[0034] The specific steps of this embodiment are as follows: (1) Obtaining positive plasmids of Shewanella putrefactive bacteria: First, log in to the NCBI database, search for and download the *Shewanella putrefactive* gyrB gene (NZ_CP066370.1). Multiple sequence alignment analysis was performed, and the results are as follows: Figure 5 As shown ( Figure 5 (The image is large and has been split into multiple groups for presentation). It was constructed into the vector pcDNA3.1. The Shewanella putrefactive bacteria positive plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and used as a standard for subsequent primer and probe screening and sensitivity evaluation.

[0035] (2) Based on the conserved region sequence of the gyrB gene of Shewanella putrefactive bacteria, five sets of candidate primer-probe combinations were designed. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The 5' end of the probe was labeled with the ROX fluorescent group and the 3' end was labeled with the BHQ2 quenching group, as shown in Table 1.

[0036] Table 1 Primer and probe sequences

[0037] (3) Primer and probe fluorescence quantitative PCR screening: Using the gyrB positive plasmid as a template, real-time quantitative PCR amplification was performed using five sets of candidate primers and probes. The following reagents were used: 10 μL of 2×Taq PCR Master Mix, 0.5 μL each of the following: fluorescent probe P (10 μM stock solution), upstream primer stock solution (10 μM), and downstream primer stock solution (10 μM), and 1×10⁻⁶ gyrB plasmid. 7 1 μL of copies / μL was added to 20 μL with sterile ddH2O. The reaction program was 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing / extension for 34 s, for 40 cycles.

[0038] The filtering results are as follows Figure 1 As shown, Figure 1 In A and B, 1 represents upstream primer F1 and downstream primer R1, 2 represents upstream primer F2 and downstream primer R2, 3 represents upstream primer F3 and downstream primer R3, 4 represents upstream primer F4 and downstream primer R4, and 5 represents upstream primer F5 and downstream primer R5. Figure 1 In the C1 array, 1 represents upstream primer F1, downstream primer R1, and probe P1; 2 represents upstream primer F2, downstream primer R2, and probe P2; 3 represents upstream primer F3, downstream primer R3, and probe P3; and 4 represents upstream primer F5, downstream primer R5, and probe P5. The detection results are obtained by comprehensively comparing the Ct value, fluorescence intensity, and amplification curve slope. Figure 1 As shown, Figure 1 A represents the primer screening results for gyrB, with Ct values ​​of 1 to 5 being 26.2, 27.93, 25.02, 25.1, and 27.34, respectively. Figure 1 C represents the probe screening results for gyrB. The Ct values ​​for 1 to 4 are 27.3, 27.24, 27.47, and 26.1, respectively. The combination F3 / R3 / P3 exhibits the highest amplification efficiency and the strongest fluorescence signal response, and the negative control shows no non-specific amplification. Therefore, F3 / R3 / P3 is determined to be the optimal primer-probe combination of this invention.

[0039] Example 2 A standard curve was established, and the concentration of plasmids containing the *Shewanella putrefactiveis* gyrB gene was determined using an ultra-micro spectrophotometer. The corresponding copy number was then calculated using the following formula: copies / μL = (ng / μL × 10⁻⁶) -9 ) × (6.022 × 10 23 ) / (DNAlength×660), prepare 1×10 6 ~1×10 2 Five concentration gradients of standards (copies / μL) were used as templates for quantitative real-time PCR amplification using F3 / R3 / P3 primer-probe combinations. A standard curve was plotted with the logarithm of the initial template copy number (lgcopies) on the x-axis and the measured Ct value on the y-axis.

[0040] The results are as follows Figure 2 As shown, Figure 2 In the middle B, 1 is 1 × 10 6 The detection rate of copies / μL plasmid was 2, which was 1×10. 5 The detection rate of copies / μL plasmids was 3, which was 1×10. 4 The detection rate of copies / μL plasmids was 4, which is 1×10⁴. 3 The detection rate of copies / μL plasmids is 5, which is 1×10. 2 The standard curve for the detection of copies / μL plasmid showed good linearity, with R0. 2 The concentration >0.99 and the amplification efficiency between 90% and 110% indicate that the F3 / R3 / P3 primer-probe combination has good amplification linearity and amplification efficiency within the target concentration range.

[0041] Example 3 To determine the detection limit, the positive plasmid was diluted to 1×10⁻⁶. 1 Two low concentration levels, 1 copy / μL and 1 copy / μL, were used for quantitative real-time PCR amplification, with 8 replicate wells for each concentration.

[0042] The results are as follows Figure 3 As shown, Figure 3 1 is 1×10 1 copies / μL, 2 is 1copies / μL, 1×10 1 Specific amplification signals were detected in all replicate wells at the copies / μL concentration level, while the detection rate at the 1 copies / μL level was less than 95%. Based on internationally accepted detection limit criteria, the detection limit for the gyrB gene of *Shewanella putrefactivee* was determined to be 10-1. 1 copies / μL.

[0043] Example 4 This embodiment provides a kit for detecting Shewanella putrefactive bacteria based on real-time quantitative PCR, including the primer-probe combination, reaction system, and reaction procedure provided in Example 1. This embodiment verifies the application and evaluates the specificity of the detection kit, and the specific operations are as follows: (1) Preparation of DNA from the test strain: Eight common pathogenic bacteria, including Shewanella putrefactiveis, Aeromonas vera, Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium psychrophilum, Aeromonas temperate, Staphylococcus aureus, and Escherichia coli, were selected and inoculated onto their respective culture media and cultured overnight. Genomic DNA was then extracted from each strain using a bacterial genomic DNA extraction kit, measured using a micro spectrophotometer, and uniformly diluted to 1×10⁻⁶. 3pg / μL was prepared for use, and gyrB plasmid standard was used as a positive control and ddH2O as a negative control to evaluate the specificity of the kit. (2) Using the genomic DNA of 8 tested strains as templates, Shewanella putrefactiveis as the positive target bacterium, gyrB positive plasmid as the positive control, and ddH2O as the negative control, real-time quantitative PCR amplification was performed using the F3 / R3 / P3 primer-probe combination. After amplification, a portion of the products was taken for 1.5% agarose gel electrophoresis verification. The results are as follows: Figure 4 As shown in A, Figure 4 In section A, 1 is the negative control ddH2O, 2 is the positive control standard plasmid, 3 is *Shewanella putrefactiveis*, 4 is *Aeromonas velutipes*, 5 is *Aeromonas hydrophila*, 6 is *Edwards tarda*, 7 is *Flavobacterium psychrophilum*, 8 is *Aeromonas sobrio*, 9 is *Staphylococcus aureus*, and 10 is *Escherichia coli*. Only the positive plasmid standard and *Shewanella putrefactiveis* showed fluorescence amplification curves. *Aeromonas velutipes*, *Aeromonas hydrophila*, *Edwards tarda*, *Flavobacterium psychrophilum*, *Aeromonas sobrio*, *Staphylococcus aureus*, *Escherichia coli*, and the negative control showed no fluorescence amplification curves. The agarose gel electrophoresis results are as follows: Figure 4 As shown in B, Figure 4 In the formula B, M stands for Marker, 1 is the negative control ddH2O, 2 is the positive control standard plasmid, 3 is Shewanella putrefactiveis, 4 is Aeromonas verrucosa, 5 is Aeromonas hydrophila, 6 is Edwardsiella tarda, 7 is Flavobacterium psychrophilum, 8 is Aeromonas temperate, 9 is Staphylococcus aureus, and 10 is Escherichia coli. The results are completely consistent with the fluorescence curve, with specific bands of the expected size appearing only in the Shewanella putrefactiveis and positive control lanes. These results indicate that the primer-probe combination described in this invention has excellent detection specificity against Shewanella putrefactiveis and exhibits no cross-reactivity with common associated and contaminating bacteria in aquaculture environments. This demonstrates that the primer-probe combination contained in this invention has strong specificity and can specifically detect Shewanella putrefactiveis.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical methods of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical methods of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical methods to deviate from the spirit and scope of the technical methods of the present invention.

Claims

1. A primer probe for detecting pathogenic Shewanella putrefaciens, characterized by comprising a primer having a nucleotide sequence represented by SEQ ID NO: 1 and a probe having a nucleotide sequence represented by SEQ ID NO:

2. The primer probe consists of any one of the primer probes from (1) to (5): (1) The upstream primer sequence is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:6, and the fluorescent probe sequence is shown in SEQ ID NO:11; (2) The upstream primer sequence is shown in SEQ ID NO:2, the downstream primer sequence is shown in SEQ ID NO:7, and the fluorescent probe sequence is shown in SEQ ID NO:12; (3) The upstream primer sequence is shown in SEQ ID NO:3, the downstream primer sequence is shown in SEQ ID NO:8, and the fluorescent probe sequence is shown in SEQ ID NO:13; (4) The upstream primer sequence is shown in SEQ ID NO:4, the downstream primer sequence is shown in SEQ ID NO:9, and the fluorescent probe sequence is shown in SEQ ID NO:14; (5) The upstream primer sequence is shown in SEQ ID NO:5, the downstream primer sequence is shown in SEQ ID NO:10, and the fluorescent probe sequence is shown in SEQ ID NO:

15.

2. The primer probe for detecting pathogenic Shewanella putrefaciens according to claim 1, wherein, The probe sequence is labeled with a ROX fluorescent group at the 5' end and a BHQ2 quencher group at the 3' end.

3. The use of a primer probe as described in any one of claims 1 or 2 in the preparation of a product for detecting pathogenic Shewanella putrefactive bacteria.

4. Use according to claim 3, characterized in that, The product is a reagent or kit.

5. Use according to claim 3, characterized in that, Application methods include: S1. Take the sample to be tested and extract DNA; S2. Using the extracted DNA as a template, perform real-time PCR using primer and probe combinations; S3. Result determination: If a fluorescent signal appears, the sample is positive, indicating the presence of Shewanella putrefactive bacteria in the sample.

6. Use according to claim 5, characterized in that, The reaction system for real-time PCR in S2 includes 10 μL of 2×TaqPCR Master Mix, 0.5 μL each of fluorescent probe, upstream primer and downstream primer, 1 μL of DNA template to be tested, and 7.5 μL of sterile ddH2O.

7. Use according to claim 5, characterized in that, The reaction program for real-time PCR was 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing / extension for 34 s, for 40 cycles.

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