Multiple MIRA primer group for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi and application of multiple MIRA primer group

Abstract

The invention discloses a multiple MIRA primer group capable of simultaneously detecting NNV, RSIV, S. iniae and V. harveyi and application of the multiple MIRA primer group, and belongs to the technical field of molecular biological detection. The invention designs four pairs of specific MIRA primers which respectively target specific genes of four pathogens of nervous necrosis virus, iridovirus, streptococcus iniae and vibrio harveyi, and provides a kit containing the primer group. According to the invention, a multiple MIRA detection method can be established, the method can be completed by reacting for 30 minutes at the constant temperature of 41 DEG C, a complicated thermal cycler is not needed, one-tube synchronous detection of DNA and RNA pathogens can be realized, and an independent reverse transcription step is not needed. The method has the advantages of rapidness, simplicity and convenience in operation, high specificity and high sensitivity, and is particularly suitable for on-site rapid screening, monitoring and early diagnosis of the important diseases in scenes such as aquaculture farms and ports.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and specifically discloses a method for simultaneously detecting NNV, RSIV, and S. iniae and V. harveyi Multiple MIRA primer sets and their applications. Background Technology

[0002] Nervous necrosis virus (NV) belongs to the Nodaviridae family ( Nodaviridae ), β Nodavirus ( Betanodavirus NNV is a viral pathogen in fish that causes viral neuronecrosis. The virus's genome consists of two single-stranded positive-sense RNAs, RNA1 and RNA2. RNA1 encodes an RNA polymerase, while RNA2 encodes a species-specific capsid protein. Based on RNA2 sequence homology, NNV can be classified into five genotypes: Red-spotted grouper neuronecrosis virus (RGNNV), turbot neuronecrosis virus (TNNV), redfin pufferfish neuronecrosis virus (TPNNV), scad neuronecrosis virus (SJNNV), and spotted flounder neuronecrosis virus (BFNNV). Due to the specificity of the RNA2 sequence in different viral species, designing primers targeting the RNA2 single-stranded sequence became the primary method for early detection of NNV.

[0003] Red sea bream iridovirus (RSIV) is an important pathogen in the aquaculture industry. RSIV mainly causes spleen and kidney enlargement and liver whitening, which seriously affects the survival rate of farmed fish. RSIV is also characterized by its latent nature and high mortality rate, making it difficult to control clinically.

[0004] Dolphin Streptococcus ( Streptococcus iniae, S. iniae It belongs to the order Lactobacilli, family Streptococci, genus Streptococcus, and is a Gram-positive bacterium. It is one of the important pathogenic bacteria of aquatic animals, possessing broad host adaptability. Its host range includes dozens of freshwater and saltwater fish species such as tilapia, rainbow trout, turbot, and flounder, making it one of the important pathogenic bacteria causing bacterial infectious diseases in many farmed fish worldwide. Regarding... S. iniae The treatment methods currently available for S. iniae The prevention and control of [the disease] still mainly relies on multiple antibiotics. However, the extensive use of antibiotics not only easily leads to the development of drug resistance in pathogens but may also disrupt the ecological balance of aquaculture water bodies. Therefore, strengthening [the management of these antibiotics] is crucial. S. iniae Early prevention is of great importance.

[0005] Vibrio harveyi ( Vibrio harveyi, V. harveyiVibrio spp. is a major pathogenic Vibrio species found in many farmed aquatic animals, widely distributed in aquaculture environments and on the surface and inside the bodies of various aquatic animals. In recent years, V. harveyi Frequent outbreaks of vibriosis caused by this disease have severely impacted the development of aquaculture in my country. Studies have shown that... V. harveyi It can infect a variety of important aquatic economic animals, such as sea bream, whiteleg shrimp, sea bass, and abalone, and its host range covers fish, shrimp, and shellfish.

[0006] Traditional methods for identifying bacterial and viral pathogens are cumbersome, complex, and time-consuming. In recent years, advancements in immunology and molecular biology techniques, such as real-time quantitative PCR, LAMP, and enzyme-linked immunosorbent assays (ELISA), have limitations in detection time and sensitivity. Recombinase-mediated isothermal amplification (MIRA) is a novel nucleic acid detection technique. The reaction is carried out at a constant temperature of 25-42℃, completing in 5-30 minutes, and requires no complex temperature cycling equipment, making it suitable for rapid on-site detection. However, there are currently no reports of multiplex MIRA methods capable of simultaneously detecting the four important aquatic pathogens mentioned above. Summary of the Invention

[0007] The purpose of this invention is to provide a method that can simultaneously detect NNV, RSIV, and S. iniae and V. harveyi This invention relates to the application of primer sets and detection methods to establish a simple and effective method for the simultaneous detection of multiple fish pathogens. The technical problem this invention aims to solve is to provide a multiplex MIRA system for the simultaneous detection of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi, with the amplified products amplified and detected using agarose gel electrophoresis.

[0008] This invention is achieved through the following technical solution: One of the objectives of this invention is to provide a method for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi The multiple MIRA primer set, the sequences of which are shown in SEQ ID NO.1-8.

[0009] Specifically, the primer set consists of the following primers: NNV-RNA2-F (SEQ ID NO.1): 5'-TTCCTGCCTGATTCAACTGACAA CG ATCACAC-3', NNV-RNA2-R (SEQ ID NO.2): 5'-GGAACCTTGTGTCATGA TG GGAGCGGTTGTCT-3'; RSIV-MCP-F (SEQ ID NO.3): 5'-TAATGTGTGGCTGCGTGTTAAGATCCCCTC CA-3', RSIV-MCP-R (SEQ ID NO.4): 5'-CGCACCTCATTGTATGGCAGAGACA CAGTAGG-3'; S. iniae- simA-F (SEQ ID NO.5): 5'-TAAAGCATTAGAAGCGGCT AAGAAAGAAG-3', S. iniae- simA-R (SEQ ID NO. 6): 5'-CAATAGTTGCTTCA AGTTCTGCTTTTCA-3'; V. harveyi- toxR-F (SEQ ID NO.7): 5'-GAGACAAAAGCAGAAACAGCCGTCGA ACAAGC-3', V. harveyi- toxR-R (SEQ ID NO. 8): 5'-TAGCATTAACACGCCAAC AGGAAGTAGCAGGG-3'.

[0010] The second objective of this invention is to provide a method for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi A multiplex MIRA detection kit, wherein the kit contains the aforementioned primer set.

[0011] Furthermore, the kit also contains buffer, enzyme premix, dNTPs, and enzyme-free water required for the MIRA isothermal amplification reaction.

[0012] The third objective of this invention is to provide a method for simultaneously detecting NNV, RSIV, and [other viruses] using the primer set or the kit. S. iniae and V. harveyi A quadruple MIRA detection method, which is not for disease treatment or prevention, includes the following steps: Step 1. Extract total nucleic acid from the sample to be tested; Step 2. Establish a quadruple MIRA reaction system: The reaction system has a total volume of 50 μL and includes: 4 μL of the template to be tested, 29.4 μL of conventional MIRA reaction buffer A, 1 μL each of forward and reverse primers, 2.5 μL of conventional MIRA reaction buffer B, and the remainder is enzyme-free water; Step 3. Isothermal amplification reaction: React the reaction system from step 2 at 39-43℃ for 25-30 min; Step 4. Result Analysis: The amplification products from Step 3 were analyzed by agarose gel electrophoresis. If specific bands appeared at positions of 303 bp, 357 bp, 239 bp, and 141 bp, they indicated the presence of nerve necrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi, respectively.

[0013] Furthermore, in step 3, the isothermal amplification reaction is carried out at a temperature of 41°C for 30 min.

[0014] Furthermore, in step 2, the final concentration of each primer in the four primer pairs mixture is 0.2 μM.

[0015] Furthermore, in step 1, the sample to be tested includes fish tissue, water samples, or samples from the aquaculture environment.

[0016] The fourth objective of this invention is to provide the application of the aforementioned multiplex MIRA primer set or the aforementioned kit in the preparation of products for the simultaneous detection or auxiliary diagnosis of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi infection.

[0017] The beneficial effects of this invention compared to existing technologies are as follows: The reaction temperature and reaction time of the single MIRA reaction system of this invention were optimized, and the detection sensitivity and specificity of the multiple MIRA amplification system were experimentally tested. Sensitivity test results show that the PCR detection of *Streptococcus dolphinus*, *Vibrio harveyi*, neuronecrosis virus, and red sea bream iridovirus has a wide detection concentration range, and samples within the low concentration range can be detected. Specificity test results show that when using this method to detect *Streptococcus dolphinus*, *Vibrio harveyi*, neuronecrosis virus, red sea bream iridovirus, *Vibrio cantonensis*, *Staphylococcus aureus*, *Vibrio parahaemolyticus*, *Rotella viride*, *Staphylococcus epidermidis*, *Lactococcus gasseri*, *Bacillus mellea*, and *Streptococcus dysgalactiae*, only *Streptococcus dolphinus*, *Vibrio harveyi*, neuronecrosis virus, and red sea bream iridovirus can be amplified well, while other bacteria cannot be amplified. Therefore, this indicates that the designed MIRA primer set has excellent specificity.

[0018] The multiplex MIRA method of this invention uses four pairs of primers simultaneously. In the same reaction system containing nucleic acid templates from four different pathogens, all four primer pairs can simultaneously and specifically amplify products of the correct size without nonspecific bands. This result demonstrates that, under optimized reaction conditions, the primer set designed in this invention exhibits good compatibility in multiplex systems, with no significant interference or cross-reaction between them, thus ensuring the reliability of the multiplex detection. Attached Figure Description

[0019] Figure 1 The optimized reaction time and reaction temperature for neuronecrosis virus single MIRA are shown in the figure. Figure 2 Optimized reaction time and temperature for single-cell MIRA of red sea bream iridovirus; Figure 3 The optimal reaction time and temperature for the single MIRA reaction of Streptococcus dolphinus are shown in the figure. Figure 4 The optimal reaction time and temperature for Vibrio harveyi single MIRA are shown in the figure. Figure 5 This is a schematic diagram of the results of the primer specificity test for neuronecrosis virus; M: Marker, 1: Streptococcus dolphinii DNA, 2: Vibrio harveyi DNA, 3: Neuronecrosis virus RNA, 4: Red sea bream iridovirus DNA, 5: Lactococcus glabra DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio kanehirae DNA, 11: Vibrio rotiferus DNA, 12: Bioluminescent bacillus from the mermaid family DNA, N: Negative control; Figure 6 This is a schematic diagram of the primer specificity test results for red sea bream iridovirus; M: Marker, 1: Streptococcus dolphinii DNA, 2: Vibrio harveyi DNA, 3: Necrosis virus RNA, 4: Red sea bream iridovirus DNA, 5: Lactococcus gasseri DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannis DNA, 11: Vibrio rotiferus DNA, 12: Bioluminescent bacillus of the mermaid DNA, N: Negative control; Figure 7 This is a schematic diagram of the primer specificity test results for Streptococcus dolphinus; M: Marker, 1: Streptococcus dolphinus DNA, 2: Vibrio harveyi DNA, 3: Necrosis virus RNA, 4: Red sea bream iridovirus DNA, 5: Lactococcus glabra DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannis DNA, 11: Rotiferus DNA, 12: Bioluminescent bacillus from the mermaid family DNA, N: Negative control; Figure 8 This is a schematic diagram of the primer specificity test results for Vibrio harveyi; M: Marker, 1: Streptococcus dolphinii DNA, 2: Vibrio harveyi DNA, 3: Necrolysis virus RNA, 4: Red sea bream iridovirus DNA, 5: Lactococcus grebeii DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannis DNA, 11: Rotiferus DNA, 12: Bioluminescent bacillus from the mermaid family DNA, N: Negative control; Figure 9 This is a schematic diagram showing the specificity test results of primers for neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi in a multiplex MIRA system; M: Marker, 1: Red sea bream iridovirus DNA, 2: Neuronecrosis virus RNA, 3: Dolphin streptococcus DNA, 4: Vibrio harveyi DNA, 5: Mixed DNA and RNA template of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi, 6: Negative control for lane 1, 7: Negative control for lane 2, 8: Negative control for lane 3, 9: Negative control for lane 4, 10: Negative control for lane 5; Figure 10 This is a schematic diagram illustrating the optimization of reaction time and temperature for multiple MIRA reactions. Figure 11 Schematic diagram for optimization of multiplex MIRA sensitivity assay; M: Marker 1: Template concentrations of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi are 8 ng / μL, 11 ng / μL, 7.4 ng / μL, and 21.6 ng / μL, respectively; 2: Template concentrations of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi are 12 ng / μL, 16.5 ng / μL, 11.1 ng / μL, and 32.4 ng / μL, respectively; 3: Template concentrations of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi are 16 ng / μL, 22 ng / μL, 14.8 ng / μL, and 43.2 ng / μL, respectively; 4: Template concentrations of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi are 20 ng / μL, 27.5 ng / μL, 27.5 ng / μL, and 27.5 ng / μL, respectively. 18.5 ng / μL, 18.5 ng / μL, 54 ng / μL; 5: negative control. Detailed Implementation

[0020] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0021] Example 1 (1) Design of MIRA primers targeting the RNA2 gene of neuronecrosis virus: Based on the RNA2 gene sequence of neuronecrosis virus published in the NCBI database (GenBank: OM305086) as the target gene, primers were designed using oligo. The primer sequences are as follows: NNV-RNA2-F (SEQ ID NO.1): 5'-TTCCTGCCTGATTCAACTGACAACGATCACAC-3', NNV-RNA2-R (SEQ ID NO.2): 5'-GGAACCTTGTGTCATGATGGGAGCGGTTGTCT-3'.

[0022] (2) Design of MIRA primers targeting the MCP gene of red sea bream iridovirus: Based on the MCP gene sequence of red sea bream iridovirus published in the NCBI database (GenBank: AY532612.1) as the target gene, primers were designed using oligo. The primer sequences are as follows: RSIV-MCP-F (SEQ ID NO. 3): 5'-TAATGTGTGGCTGCGTGTTAAGATCCCC TCCA-3'; RSIV-MCP-R (SEQ ID NO. 4): 5'-CGCACCTCATTGTATGGCAGA GACACAGTAGG-3'.

[0023] (3) Design of MIRA primers targeting the simA gene of Streptococcus dolphinus: Based on the simA gene sequence (GenBank: JF330100.1) published in the NCBI database as the target gene, primers were designed using oligo. The primer sequences are as follows: S. iniae- simA-F (SEQ ID NO.5):5'-TAAAGCATTA GAAGCGGCTA AGAAAGAAG-3', S. iniae- simA-R (SEQ ID NO. 6): 5'-CAATAGTTGCTTCAAGTTCTGCTTTTTCA-3'.

[0024] (4) Design of MIRA primers targeting the toxR gene of Vibrio harveyi: Based on the toxR gene sequence (GenBank: DQ640257.1) published in the NCBI database as the target gene, primers were designed using oligo. The primer sequences are as follows: V. harveyi- toxR-F (SEQ ID NO.7):5'-GAGACAA AAGCAGAAACAG CCGTCGAACAAGC-3', V. harveyi-toxR-R (SEQ ID NO.8):5'-TAGCATTAACACGCCACAGGAAGTAGCAGGG-3'; The primers mentioned above were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0025] Example 2 DNA and RNA were extracted from Streptococcus dolphinii, Vibrio harveyi, neuronecrosis virus, and red sea bream iridovirus using commercially available kits.

[0026] (1) The system optimization of the MIRA detection method for neuronecrosis virus is as follows: 4 ng / uL and 40 pg / uL of neuronecrosis virus RNA template are used for the experiment. 7.35 μL of MIRA kit A Buffer (Anpu Future Biotechnology Co., Ltd., conventional MIRA reaction buffer), 1 μL each of the above upstream and downstream primers (concentration of 0.8 um), 1.25 uL of B Buffer (Anpu Future Biotechnology Co., Ltd., conventional MIRA reaction buffer), and enzyme-free water are added to make up to 12.5 μL for MIRA amplification. Reaction conditions: Temperature gradients of 39℃, 41℃, and 43℃ were used for experiments, with reaction times of 5 min and 10 min, respectively. The MIRA amplification product was purified by adding 150 μL of DNA extraction phenol, centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 μL of 6× loading buffer was added and mixed well, and 5 μL was used for agarose gel electrophoresis (135 V, 20 min). The target band for neuronecrosis virus was 303 bp. It can be seen that... Figure 1 Under the condition of reacting at 43℃ for 10 min, the target band can be amplified with 4 ng / μL template, but no target band appears with 40 pg / μL template. Therefore, the limit of detection of NNV is 4 ng / μL.

[0027] (2) The optimized system for the detection method of red sea bream iridovirus MIRA was as follows: 5.5 ng / uL and 55 pg / uL of red sea bream iridovirus DNA template were used for the experiment; 7.35 μL of MIRA kit A Buffer, 1 μL each of the above upstream and downstream primers (concentration of 0.8 μm), 1.25 uL of B Buffer, and enzyme-free water were added to make up to 12.5 μL for MIRA amplification. Reaction conditions: The temperature gradient was 39℃, 41℃, and 43℃, and the reaction time was 5 min and 10 min, respectively. The MIRA amplification product was purified by adding 150 uL of DNA extraction phenol, centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 uL of 6× loading buffer was added and mixed well. 5 uL was taken for agarose gel electrophoresis (135 V, 20 min). The target band of red sea bream iridovirus was 357 bp. Figure 2 At temperatures of 41℃ and 43℃ for 5 min and 10 min respectively, the target band could be amplified with a template of 5.5 ng / uL, but no target band was observed with a template of 55 pg / uL. Therefore, the detection limit of single MIRA for red sea bream iridovirus is 5.5 ng / uL.

[0028] (3) The optimized system for the MIRA detection method of Streptococcus dolphin virus was as follows: 3.7 ng / uL and 37 pg / uL of Streptococcus dolphin DNA template were used for the experiment. 7.35 μL of MIRA kit A Buffer, 1 μL each of the above upstream and downstream primers (concentration of 0.8 μm), 1.25 uL of B Buffer, and enzyme-free water were added to make up to 12.5 μL for MIRA amplification. Reaction conditions: The temperature gradient was 39℃, 41℃, and 43℃, and the reaction time was 5 min and 10 min, respectively. The MIRA amplification product was purified by adding 150 uL of DNA extraction phenol, centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 uL of 6× loading buffer was added and mixed well. 5 uL was taken for agarose gel electrophoresis (135 V, 20 min). The target band of Streptococcus dolphin was 239 bp. Figure 3 At temperatures of 39℃, 41℃, and 43℃, and reaction times of 5 min and 10 min, respectively, the target band could be amplified with a template concentration of 3.7 ng / uL, while no target band was observed with a template concentration of 37 pg / uL. Therefore, the detection limit of Streptococcus dolphin single MIRA is 3.7 ng / uL.

[0029] (4) The optimized system for the MIRA detection method of Vibrio harveyi was as follows: Vibrio harveyi DNA template was prepared at 10.8 ng / uL and 108 pg / uL. MIRA kit A Buffer 7.35 μL, upstream and downstream primers 1 μL each (concentration 0.8 μm), B Buffer 1.25 μL, and enzyme-free water was added to bring the total volume to 12.5 μL for MIRA amplification. Reaction conditions: Temperature gradients were set at 39℃, 41℃, and 43℃, with reaction times of 5 min and 10 min, respectively. The MIRA amplification product was purified by adding 150 μL of DNA extraction phenol, centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 μL of 6× loading buffer was added and mixed well. 5 μL was then subjected to agarose gel electrophoresis (135 V, 20 min). The target band of Vibrio harveyi was 141 bp. Figure 4 At temperatures of 39℃, 41℃, and 43℃, and reaction times of 5 min and 10 min, the target band could be amplified with a template concentration of 10.8 ng / uL, while no target band was observed with a template concentration of 108 pg / uL. Therefore, the detection limit of Vibrio harveyi single MIRA is 10.8 ng / uL.

[0030] Example 3 The above-mentioned neural necrosis virus (NNV)-RNA2-F / R primers were used to simultaneously perform specific detection on 12 marine pathogenic bacteria (Table 1), and the specificity of the amplification primers was evaluated: Table 1. Strains used for PCR primer specificity analysis ; Using DNA or RNA from 12 bacteria and viruses—including *Streptococcus dolphinus*, *Vibrio harveyi*, neuronecrosis virus, *Red sea bream iridovirus*, *Vibrio cannis*, *Vibrio parahaemolyticus*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Lactococcus gasseri*, *Vibrio rotiferus*, *Bacillus melanogaster*, and *Streptococcus dysgalactiae*—as templates, and using NNV-RNA2-F / R of neuronecrosis virus as primers, a total reaction system of 12.5 μL was constructed. MIRA amplification was performed using 7.35 μL of MIRA kit A buffer, 1 μL each of forward and reverse primers (0.8 μm concentration), 1.25 μL of B buffer, and enzyme-free water to a final volume of 12.5 μL. Using the optimized reaction conditions described above, the MIRA amplification product was purified by adding 150 μL of DNA extraction phenol. The product was centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 μL of 6× loading buffer was added and mixed well. 5 μL of the supernatant was then subjected to agarose gel electrophoresis (135 V, 20 min). The results of primer specificity verification are shown in the figure below. Figure 5As shown, except for the neural necrosis virus which amplified the target band (positive), the results of the other 11 pathogens and the negative control were all negative, indicating that the NNV-RNA2-F / R amplification primers of the present invention have good specificity.

[0031] The RSIV-MCP-F / R primers for red sea bream iridovirus were used to simultaneously detect the specificity of 12 marine pathogens (Table 1). The specificity of the amplification primers was evaluated. Using DNA or RNA of 12 bacteria and viruses (including *Streptococcus dolphinii*, *Vibrio harveyi*, neuronecrosis virus, red sea bream iridovirus, *Vibrio cannis*, *Vibrio parahaemolyticus*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Lactococcus gasseri*, *Vibrio rotiferus*, *Bacillus spp.*, and *Streptococcus faecium*) as templates, and the RSIV-MCP-F / R primers for red sea bream iridovirus as primers, a total reaction system of 12.5 μL was constructed. MIRA amplification was performed using 7.35 μL of MIRA kit A buffer, 1 μL each of forward and reverse primers (0.8 μm concentration), 1.25 μL of B buffer, and enzyme-free water to a final volume of 12.5 μL. Using the optimized reaction conditions described above, the MIRA amplification product was purified by adding 150 μL of DNA extraction phenol. The product was centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 μL of 6× loading buffer was added and mixed well. 5 μL of the supernatant was then subjected to agarose gel electrophoresis (135 V, 20 min). The results of primer specificity verification are shown in the figure below. Figure 6 As shown, except for the red sea bream iridovirus which amplified the target band (positive), the results of the other 11 pathogens and the negative control were all negative, indicating that the RSIV-MCP-F / R amplification primers of the present invention have good specificity.

[0032] Using the above-mentioned Streptococcus dolphin S. iniae- SimA-F / R primers were used to simultaneously and specifically detect 12 marine pathogenic bacteria (Table 1). The specificity of the amplification primers was evaluated: DNA or RNA of 12 bacteria and viruses, including *Streptococcus dolphinus*, *Vibrio harveyi*, neuronecrosis virus, red sea bream iridovirus, *Vibrio cannis*, *Vibrio parahaemolyticus*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Lactococcus gasseri*, *Vibrio rotiferus*, *Bacillus luminifera*, and *Streptococcus dysgalactiae*, were used as templates. S. iniae-Using simA-F / R as primers, a total reaction volume of 12.5 μL was constructed. The MIRA kit contained 7.35 μL of A Buffer, 1 μL each of forward and reverse primers (0.8 μm concentration), 1.25 μL of B Buffer, and enzyme-free water to a final volume of 12.5 μL for MIRA amplification. Under the optimized reaction conditions, the MIRA amplification product was purified by adding 150 μL of DNA extraction phenol, centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 μL of 6× loading buffer was added and mixed well. 5 μL of the mixture was then subjected to agarose gel electrophoresis (135 V, 20 min). The results of primer specificity verification are shown in the figure below. Figure 7 As shown, except for *Streptococcus dolphinii* which amplified the target band (positive), the results of the other 11 pathogen strains and the negative control were all negative, indicating that the present invention... S. iniae- The simA-F / R amplification primers have good specificity.

[0033] Using the above-mentioned Vibrio harveyi V. harveyi- The toxR-F / R primers were used to simultaneously and specifically detect 12 marine pathogenic bacteria (Table 1). The specificity of the amplification primers was evaluated: DNA or RNA from 11 bacteria and viruses, including *Streptococcus dolphinii*, *Vibrio harveyi*, neuronecrosis virus, red sea bream iridovirus, *Vibrio kanehirae*, *Vibrio parahaemolyticus*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Lactococcus gasseri*, *Vibrio rotiferus*, *Bacillus luminifera*, and *Streptococcus dysgalactiae*, were used as templates. V. harveyi- Using toxR-F / R as primers, a total reaction volume of 12.5 μL was constructed. The MIRA kit contained 7.35 μL of A Buffer, 1 μL each of forward and reverse primers (0.8 μm concentration), 1.25 μL of B Buffer, and enzyme-free water to a final volume of 12.5 μL for MIRA amplification. Using the optimized reaction conditions, the MIRA amplification product was purified by adding 150 μL of DNA extraction phenol, centrifuged at 12000 rpm for 120 s, and the supernatant was collected. 2.5 μL of 6× loading buffer was added and mixed well. 5 μL of the mixture was then subjected to agarose gel electrophoresis (135 V, 20 min). The results of primer specificity verification are shown in the figure below. Figure 8 As shown, except for Vibrio harveyi, which amplified the target band (positive), the results of the other 11 pathogen strains and the negative control were all negative, indicating that the present invention... V. harveyi- The toxR-F / R amplification primers have good specificity.

[0034] Example 4 DNA or RNA from Streptococcus dolphinii, Vibrio harveyi, neuronecrosis virus, and red sea bream iridovirus were selected as templates, as well as mixtures thereof. NNV-RNA2, RSIV-MCP, and other methods were used to develop the templates. S. iniae- simA、 V. harveyi- Amplification and detection were performed using MIRA primers for toxR, and the results were as follows: Figure 9 The display shows RSIV, NNV, S. iniae and V. harveyi All primer pairs amplified their respective target bands, while the corresponding negative control showed no band, indicating that the designed four primer pairs have good specificity and can be used for quadruple MIRA detection experiments. Furthermore, as... Figure 9 As shown, all four MIRA experiments can amplify RSIV, NNV, and S. iniae and V. harveyi The target band. Additionally, such as Figure 9 As shown in lane 5, in the reaction system where four primer pairs coexist with four templates, the electrophoresis results only showed four specific bands (303 bp, 357 bp, 239 bp, 141 bp) corresponding to the four target pathogens. No other bands of different sizes or obvious primer dimer bands were observed, indicating that each primer pair maintained high specificity under multiplex reaction conditions and there was no cross-amplification between them.

[0035] Experimental Example 5 This example presents the optimized reaction time and temperature in a multiplex MIRA reaction system: A certain concentration of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi were selected as templates. The optimized MIRA reaction times were 25 min and 30 min, and the optimized reaction temperatures were 39℃, 41℃, and 43℃. Figure 10 As can be seen, 41℃ and 30 min of reaction in the multiple MIRA reaction system yielded better results, thus determining the optimal multiple MIRA reaction conditions.

[0036] Experimental Example 6 This experimental example demonstrates the sensitivity detection experiment in a multiplex MIRA reaction system. Under an optimized multiplex reaction system, multiplex MIRA detection experiments were conducted by serially diluting the reaction template. The corresponding template concentrations were as follows: 1: 8 ng / μL for neuronecrosis virus, iridovirus of red sea bream, Streptococcus dolphinus, and Vibrio harveyi, 11 ng / μL, 7.4 ng / μL, and 21.6 ng / μL, respectively; 2: 12 ng / μL for neuronecrosis virus, iridovirus of red sea bream, Streptococcus dolphinus, and Vibrio harveyi, 16.5 ng / μL, 11.1 ng / μL, and 32.4 ng / μL, respectively; 3: 16 ng / μL for neuronecrosis virus, iridovirus of red sea bream, Streptococcus dolphinus, and Vibrio harveyi, 22 ng / μL, 14.8 ng / μL, and 43.2 ng / μL, respectively; 4: 20 ng / μL for neuronecrosis virus, iridovirus of red sea bream, Streptococcus dolphinus, and Vibrio harveyi, 18.5 ng / μL, and 27.5 ng / μL, respectively. ng / μL, 54 ng / uL, 5: negative control; via Figure 11 As can be seen from the series of dilution tests, under the optimal reaction system, the limits of detection (LODs) of the described multiplex MIRA method for neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi were 16 ng / μL, 22 ng / μL, 14.8 ng / μL, and 43.2 ng / μL, respectively. This indicates that the multiplex MIRA detection method established in this invention can simultaneously detect neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus, and Vibrio harveyi within 30 minutes of reaction. It exhibits high detection efficiency, high specificity, and high sensitivity. Compared to PCR, it eliminates the need for large laboratory equipment, requiring only a metal bath and water bath for temperature amplification, making it more suitable for field testing and applicable to a wider range of scenarios.

[0037] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention.

Claims

1. A method for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi The multiple MIRA primer set is characterized by, The sequences of the primer set are shown in SEQ ID NO.1-8.

2. A method for simultaneously detecting NNV, RSIV, S. iniae and V. harveyi A multiplex MIRA detection kit, characterized in that, The kit contains the primer set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also contains buffer, enzyme premix, dNTPs, and enzyme-free water required for the MIRA isothermal amplification reaction.

4. A method for simultaneously detecting NNV, RSIV, and [other pathogens] using the primer set of claim 1 or the kit of claim 3. S. iniae and V. harveyi The method, which is for non-disease treatment or prevention purposes, is characterized in that... Includes the following steps: (1) Extract total nucleic acid from the sample to be tested; (2) Establishing a multiplex MIRA reaction system: The reaction system has a total volume of 50 μL and includes: 4 μL of nucleic acid template to be tested, 29.4 μL of conventional MIRA reaction buffer A, 8 μL of the mixture of primers described in claim 1, 2.5 μL of conventional MIRA reaction buffer B, and the remainder is enzyme-free water; (3) Isothermal amplification reaction: The reaction system of step (2) is reacted at 39-43℃ for 25-30 min; (4) Results analysis: The amplification products of step (3) were analyzed by agarose gel electrophoresis. If specific bands appeared at positions of 303 bp, 357 bp, 239 bp and 141 bp, they indicated the presence of nerve necrosis virus, red sea bream iridovirus, dolphin streptococcus and Vibrio harveyi, respectively.

5. The method according to claim 4, characterized in that, In step (3), the temperature of the isothermal amplification reaction is 41°C and the reaction time is 30 min.

6. The method according to claim 4, characterized in that, In step (2), the final concentration of each primer in the primer set mixture is 0.2 μM.

7. The method according to claim 4, characterized in that, In step (1), the sample to be tested includes fish tissue, water body or aquaculture environment sample.

8. The use of the multiplex MIRA primer set of claim 1 in the preparation of a product for the simultaneous detection or auxiliary diagnosis of neuronecrosis virus, red sea bream iridovirus, dolphin streptococcus and Vibrio harveyi infection.

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