Multiplex PCR primer set for simultaneous detection of four pathogens, detection method, and application

Abstract

The present invention relates to a multiplex PCR primer set for simultaneously detecting four pathogens, as well as a detection method and application thereof, belonging to the field of molecular biology. The four pathogens are neural necrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi. The primer set comprises SEQ ID NOs. 1-8. Using the primers of the present invention, neural necrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi can be simultaneously detected in a short period of time, thereby achieving the purpose of establishing a simple and effective method for simultaneously detecting multiple fish pathogens. Furthermore, the detection primer set and detection method of the present method have good specificity and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and specifically discloses a multiplex PCR primer set for simultaneously detecting four pathogens, a detection method and an application. Background Art

[0002] Nervous necrosis virus (NNV) belongs to the Nodaviridae family and the Betanodavirus genus. This genus specifically infects fish, causing viral neuronecrosis. NNV consists of two single-stranded positive-sense RNAs: RNA1, which encodes RNA polymerase, and RNA2, which encodes a species-specific capsid protein. NNV can be further divided into five types based on the homology of their RNA2 sequences: red-spotted grouper neural necrosis virus (RGNNV), turbot neural necrosis virus (TNNV), redfin pufferfish neural necrosis virus (TPNNV), yellowtail trevally neural necrosis virus (SJNNV), and striped flounder neural necrosis virus (BFNNV). Early detection of NNV often utilizes the specificity of RNA2 sequences across virus species, designing primers targeting the RNA2 single-strand sequence.

[0003] Red sea bream iridovirus (RSIV) is a major pathogen in the aquaculture industry. RSIV primarily causes enlarged spleen and kidneys and a whitish liver, seriously endangering the survival of fish. RSIV is also difficult to control clinically due to its insidious nature and high mortality rate.

[0004] Streptococcus iniae, a Gram-positive bacterium belonging to the order Lactobacilales, family Streptococcus, genus Streptococcus, is a major aquatic pathogen with a broad host range, including dozens of brackish and marine fish species such as tilapia, rainbow trout, turbot, and flounder. It has become a major cause of invasive infections and outbreaks in fish worldwide. Treatment for Streptococcus iniae primarily relies on the use of multiple antibiotics. Excessive antibiotic use not only leads to bacterial resistance but also destabilizes water bodies. Therefore, early prevention of Streptococcus iniae is crucial.

[0005] Vibrio harveyi is a major pathogen of various aquatic animals, widely distributed in marine water and within the bodies and exteriors of aquatic animals. In recent years, frequent outbreaks of vibriosis caused by Vibrio harveyi have severely impacted the development of my country's aquaculture industry. Studies have shown that the reported strains of Vibrio harveyi can infect important aquatic species, including sea bream, whiteleg shrimp, sea bass, and abalone.

[0006] Traditional methods for identifying bacteria and viruses are cumbersome, complex, and time-consuming. Recent advances in immunology and molecular biology techniques, such as real-time fluorescence quantitative PCR, LAMP, and enzyme-linked immunosorbent assay (ELISA), have limited detection time and sensitivity. In contrast, PCR offers high sensitivity, strong specificity, and ease of use. However, limited research exists on the simultaneous detection of NNV, iridovirus, Streptococcus iniae, and Vibrio harveyi. Therefore, developing a multiplex PCR assay capable of simultaneously detecting NNV, iridovirus, Streptococcus iniae, and Vibrio harveyi has significant practical significance and application value. Summary of the Invention

[0007] The present invention aims to provide a primer set and detection method capable of simultaneously detecting NNV, iridovirus, Streptococcus iniae, and Vibrio harveyi in a short period of time, thereby establishing a simple and effective method for the simultaneous detection of multiple fish pathogens. The technical problem addressed by the present invention is to provide a multiplex PCR system for the simultaneous detection of NNV, iridovirus, Streptococcus iniae, and Vibrio harveyi, with the amplified products being amplified and detected using agarose gel electrophoresis.

[0008] The present invention is achieved through the following technical solutions:

[0009] A multiplex PCR primer set for simultaneously detecting four pathogens, wherein the four pathogens are neuronecrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi, and the primer set consists of the following primers: NNV-RNA2-F: 5'-GCTAGAATCTTCCAGCGATAC-3' (SEQ ID NO. 1), NNV-RNA2-R: 5'-CTCGATCAACATCTCCAGTTC-3' (SEQ ID NO. 2);

[0010] RSIV-MCP-F: 5'-CCGCCGCCTACCTTAATTTGCC-3' (SEQ ID NO.3), RSIV-MCP-R: 5'-TTGTTGTTGACATACACGGGAC-3' (SEQ ID NO.4); simA-F: 5'-TGAAGAGCTTGACAAACTAAATG-3' (SEQ ID NO.5), simA-R: 5'-ACTTGCTGTGAAGAAAGGGTTA-3' (SEQ ID NO.6); toxR-F: 5'-ACCTAGAGCAATTCGCAGAG-3' (SEQ ID NO.7), toxR-R: 5'-TGACGAAATTGTGATTCTGC-3' (SEQ ID NO.8).

[0011] A multiplex PCR detection kit for simultaneously detecting four pathogens, comprising the primer set.

[0012] The present invention also provides a method for performing multiplex PCR detection using the primer set. The system of the PCR detection method is as follows: 1 μL of the template of the sample to be tested, 12.5 μL of PCR mix, 1 μL of each of the four pairs of upstream and downstream primers, and enzyme-free water to make up to 25 μL for PCR amplification; the reaction procedure is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 63.8°C for 30 seconds, extension at 72°C for 1 minute, amplification for 30 cycles, and storage of the amplified products at 4°C; PCR products are subjected to agarose gel electrophoresis for detection, and the target bands of neural necrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi are 544 bp, 472 bp, 325 bp, and 199 bp, respectively.

[0013] The present invention also provides an application of the primer set, which is for non-disease detection and treatment purposes. The application method is to use the PCR primer set to perform a PCR reaction, and perform agarose gel electrophoresis on the reaction products. The target bands of neural necrosis virus, iridescent virus, Streptococcus iniae and Vibrio harveyi are divided into 544bp, 472bp, 325bp and 199bp.

[0014] The present invention has the following beneficial effects compared to the prior art: The sensitivity and specificity of the multiplex PCR amplification system of the present invention were tested. The sensitivity test results showed that the sensitivity of PCR for detecting Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus has a wide detection range, and can be detected in samples within a certain range. The specificity test results showed that when this method was used to detect Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio cannulae, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio rotifer, Staphylococcus epidermidis, Lactococcus garris, Photobacterium mermanii, and Streptococcus dysgalactiae, only Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus could be amplified well, while other bacteria could not be amplified. Therefore, it shows that the primer set has good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the results of the neural necrosis virus primer specificity test; M: Marker, 1: Streptococcus iniae DNA, 2: Vibrio harveyi DNA, 3: neural necrosis virus cDNA, 4: Iridovirus DNA, 5: Lactococcus garvais DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannulatus DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control;

[0016] Figure 2 Schematic diagram of the results of the iridovirus primer specificity test; M: Marker, 1: Streptococcus iniae DNA, 2: Vibrio harveyi DNA, 3: Neuronecrosis virus cDNA, 4: iridovirus DNA, 5: Lactococcus garvais DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannulatus DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control;

[0017] Figure 3 Schematic diagram of the results of the primer specificity test for Streptococcus iniae; M: Marker, 1: Streptococcus iniae DNA, 2: Vibrio harveyi DNA, 3: Neuronecrosis virus cDNA, 4: Iridovirus DNA, 5: Lactococcus garvae DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannulata DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control;

[0018] Figure 4Schematic diagram of the primer specificity test results for Vibrio harveyi; M: Marker, 1: Streptococcus iniae DNA, 2: Vibrio harveyi DNA, 3: Neuronecrosis virus cDNA, 4: Iridovirus DNA, 5: Lactococcus garvae DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannulatus DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control;

[0019] Figure 5 Schematic diagram of the specificity test results of primers for neural necrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi in a multiplex system; M: Marker, 1: neural necrosis virus cDNA, 2: iridovirus DNA, 3: Streptococcus iniae DNA, 4: Vibrio harveyi DNA, 5: mixed DNA of neural necrosis virus, iridovirus, Streptococcus iniae, 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;

[0020] Figure 6 Schematic diagram of the results of the neuronecrosis virus sensitivity test; M: Marker, 1: 17.4 ng / μL, 2: 1.74 ng / μL, 3: 174 pg / μL, 4: 17.4 pg / μL, N: negative control;

[0021] Figure 7 Schematic diagram of the iridovirus sensitivity test results; M: Marker, 1: 20.8 ng / μL, 2: 2.08 ng / μL, 3: 208 pg / μL, 4: negative control;

[0022] Figure 8 Schematic diagram of the results of the sensitivity test of Streptococcus iniae; M: Marker, 1: 42.1 ng / μL, 2: 4.21 ng / μL, 3: 421 pg / μL, 4: negative control;

[0023] Figure 9 Schematic diagram of the Vibrio harveyi sensitivity test results; M: Marker, 1: 45.6 ng / μL, 2: 4.56 ng / μL, 3: 456 pg / μL, 4: negative control;

[0024] Figure 10 Schematic diagram of different annealing temperatures for the multiplex PCR system; M: Marker, 1: 58°C, 2: 59°C, 3: 60°C, 4: 61°C, 5: 62°C, 6: 63°C, 7: 63.8°C, N: negative control. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further explained below through examples, but the protection scope of the present invention is not limited in any form by the examples.

[0026] Example 1:

[0027] (1) Design of PCR primers targeting the RNA2 gene of neuronecrosis virus: Based on the RNA2 gene sequence of neuronecrosis virus published in the NCBI database (GenBank: OM305086), primers were designed using oligo. The designed primer sequences are as follows: NNV-F: 5'-GCTAGAATCTTCCAGCGATAC-3', NNV-R: 5'-CTCGATCAACATCTCCAGTTC-3'.

[0028] (2) Design of PCR primers targeting the MCP gene of iridovirus: Based on the MCP gene sequence of iridovirus published in the NCBI database (GenBank: AY532612.1) as the target gene, primers were designed using oligonucleotides. The designed primer sequences are as follows:

[0029] RSIV-MCP-F: 5'-CCGCCGCCTACCTTAATTTGCC-3';

[0030] RSIV-MCP-R: 5'-TTGTTGTTGACATACACGGGAC-3'.

[0031] (3) Design of PCR primers targeting the simA gene of Streptococcus dolphinus: Based on the simA gene sequence published in the NCBI database (GenBank: JF330100.1), primers were designed using oligo. The designed primer sequences are as follows: simA-F: 5'-TGAAGAGCTTGACAAACTAAATG-3',

[0032] simA-R:5'-ACTTGCTGTGAAGAAAGGGTTA-3'.

[0033] (4) Design of PCR primers targeting the toxR gene of Vibrio harveyi: Based on the toxR gene sequence published in the NCBI database (GenBank: DQ640257.1) as the target gene, primers were designed using oligo. The designed primer sequences are as follows: toxR-F: 5'-ACCTAGAGCAATTCGCAGAG-3',

[0034] toxR-R:5'-TGACGAAATTGTGATTCTGC-3'.

[0035] The above primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0036] Example 2

[0037] For the extraction of DNA and cDNA of Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus, 1 ml of bacterial solution was added to a 1.5 ml EP tube and extracted using a commercial bacterial DNA kit. Neuronecrosis virus was extracted using trizol, and iridovirus was extracted using phenol / chloroform.

[0038] The NNV-RNA2 primers were used to simultaneously perform specific detection on 12 marine pathogens (Table 1), and the specificity of the amplification primers was analyzed and evaluated:

[0039] Table 1 Strains used for PCR primer specificity analysis

[0040] .

[0041] DNA or cDNA of 12 bacteria and viruses, including Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campestris, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garneri, Vibrio rotiferus, Photobacterium mermanii, and Streptococcus dysgalactiae, was used as template, and NNV-RNA2 of neuronecrosis virus was used as primer to construct a 20 μL reaction system. Each system consisted of 1 μL of upper and lower primers, 10 μL of mix, 1 μL of template, and 7 μL of DEPC water. The reaction was pre-denatured at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. 30 cycles of amplification were performed, and the amplified products were stored at 4°C. 5 μL of PCR product was tested by 1.5% agarose gel electrophoresis. The results of primer specificity verification are shown in the figure. Figure 1 As shown, except for the neuronecrosis virus which had an amplification band and was positive, the results of the other 11 pathogens and the negative control were all negative, indicating that the NNV-RNA2 amplification primers of the present invention have good specificity.

[0042] The RSIC-MCP primers for iridovirus were used to simultaneously detect 12 marine pathogens (Table 1). The specificity of the amplification primers was evaluated. DNA or cDNA from 12 bacteria and viruses, including Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campestris, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garneri, Vibrio rotiferus, Photobacterium mermanii, and Streptococcus dysgalactiae, was used as template. A 20 μL reaction system was constructed using the RSIV-MCP primers for iridovirus. Each reaction system consisted of 1 μL of each upper and lower primer, 10 μL of the mix, 1 μL of template, and 7 μL of DEPC water. The reaction was pre-denatured at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 64°C for 30 seconds, and extension at 72°C for 1 minute. The amplified products were stored at 4°C. A 5 μL sample of PCR product was analyzed by electrophoresis on a 1.5% agarose gel. The specificity verification results of the amplification primers are as follows Figure 2 As shown, except for the iridovirus which had an amplified band and was positive, the results of the other 11 pathogens and the negative control were all negative, indicating that the RSIV-MCP amplification primers of the present invention have good specificity.

[0043] The above-mentioned Sima primers of Streptococcus iniae were used to simultaneously perform specific detection on 12 marine pathogens (Table 1), and the specificity of the amplification primers was analyzed and evaluated: DNA or cDNA of 12 bacteria and viruses such as Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campestris, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garneri, Vibrio rotiferus, Photobacterium mermaidii, and Streptococcus dysgalactiae were used as templates, and Sima of Streptococcus iniae was used as primers to construct a 20 μL reaction system. Each system consisted of 1 μL of the upper and lower primers, 10 μL of mix, 1 μL of template, 7 μL of DEPC water, and pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 1 minute, and 30 cycles of amplification. The amplified products were stored at 4°C. 5 μL of PCR product was taken for detection by 1.5% agarose gel electrophoresis. The specificity verification results of the amplification primers are shown as follows. Figure 3 As shown, except for Streptococcus iniae, which showed an amplification band and was positive, the results of the other 11 pathogens and the negative control were all negative, indicating that the simA amplification primers of the present invention have good specificity.

[0044] The aforementioned V. harveyi toxR primers were used to simultaneously detect 12 marine pathogens (Table 1). The specificity of the amplification primers was evaluated. DNA or cDNA from 11 bacteria and viruses, including Streptococcus hiveii, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campestris, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garvei, Vibrio rotiferus, Photobacterium mermanii, and Streptococcus dysgalactiae, was used as template. A 20 μL reaction system was constructed using the V. harveyi toxR primers. Each system consisted of 1 μL of each upper and lower primer, 10 μL of the mix, 1 μL of template, and 7 μL of DEPC water. The reaction was pre-denatured at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 1 min. The amplified products were stored at 4°C. A 5 μL sample of PCR product was analyzed by electrophoresis on a 1.5% agarose gel. The specificity verification results of the amplification primers are as follows Figure 4 As shown, except for Vibrio harveyi which showed an amplified band and was positive, the results of the other 11 pathogens and the negative control were all negative, indicating that the toxR amplification primers of the present invention have good specificity.

[0045] DNA or cDNA of Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus were selected as templates and amplified using primers simA, toxR, NNV-RNA2, and RSIV-MCP. The results were as follows: Figure 5 The results showed that Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus were amplified and detected. This indicates that the four primer pairs are highly specific and can be used for the simultaneous detection of neuronecrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi in a multiplex PCR reaction system.

[0046] Example 3

[0047] This example is about establishing sensitivity: the initial concentration of 17.4 ng / μL of the neural necrosis virus was selected, and it was diluted according to a ten-fold gradient to measure the single-plex sensitivity. The concentrations of the neural necrosis virus cDNA were 17.4 ng / μL, 1.74 ng / μL, 174 pg / μL, and 17.4 pg / μL.

[0048] A PCR reaction system was established with 1 μL of cDNA from each concentration of NNV virus, 10 μL of PCR mix, 1 μL of upstream and downstream primers, and enzyme-free water to 20 μL for PCR amplification. The reaction procedure was as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. The amplified product was stored at 4°C. 5 μL of the PCR product was analyzed by electrophoresis on a 1.5% agarose gel.

[0049] Sensitivity test of neuronecrosis virus Figure 6 As shown, the minimum detection limit was 1.74 ng / μL.

[0050] An iridescent virus with an initial concentration of 20.8 ng / μL was selected and diluted in a ten-fold gradient to measure the single-dose sensitivity. The concentrations of iridescent virus DNA were 20.8 ng / μL, 2.08 ng / μL, and 208 pg / μL, respectively.

[0051] A PCR reaction system was established with 1 μL of each iridovirus DNA concentration, 10 μL of PCR mix, 1 μL of upstream and downstream primers, and enzyme-free water to 20 μL for PCR amplification. The reaction procedure was as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 64°C for 30 seconds, and extension at 72°C for 1 minute. The amplified product was stored at 4°C. 5 μL of the PCR product was analyzed by electrophoresis on a 1.5% agarose gel.

[0052] Sensitivity test for iridovirus Figure 7 As shown, the minimum detection limit was 2.08 ng / μL.

[0053] The initial concentration of Streptococcus iniae was 42.1 ng / μL, and it was diluted in a ten-fold gradient to measure the single sensitivity. The concentrations of Streptococcus iniae DNA were 42.1 ng / μL, 4.21 ng / μL, and 421 pg / μL, respectively.

[0054] A PCR reaction system was established with 1 μL of each Streptococcus iniae DNA concentration, 10 μL of PCR mix, 1 μL of upstream and downstream primers, and enzyme-free water to 20 μL for PCR amplification. The reaction procedure was as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1 minute. The amplified product was stored at 4°C. 5 μL of the PCR product was analyzed by electrophoresis on a 1.5% agarose gel.

[0055] Susceptibility test of Streptococcus iniae Figure 6 As shown, the minimum detection limit was 4.21 ng / μL.

[0056] An initial concentration of 45.6 ng / μL of Vibrio harveyi was selected and diluted in a ten-fold gradient to measure the single-plex sensitivity. The concentrations of DNA of Vibrio harveyi were 45.6 ng / μL, 4.56 ng / μL, and 456 pg / μL, respectively.

[0057] A PCR reaction system was established with 1 μL of DNA from each concentration of Vibrio harveyi, 10 μL of PCR mix, 1 μL of upstream and downstream primers, and enzyme-free water to 20 μL for PCR amplification. The reaction procedure was as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, and extension at 72°C for 1 minute. The amplified product was stored at 4°C. 5 μL of the PCR product was analyzed by electrophoresis on a 1.5% agarose gel.

[0058] Vibrio harveyi susceptibility test Figure 7 As shown, the minimum detection limit was 4.56 ng / μL.

[0059] The minimum detection limits of Streptococcus iniae, Vibrio harveyi, neuronecrosis virus and iridovirus were tested at different temperatures in a multiplex PCR system.

[0060] The minimum detection limit of an equal mixture of Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus.

[0061] A multiplex PCR reaction system was established: a mixture of four DNA and cDNA templates, 12.5 μL of PCR mix, 1 μL of each of the four upstream and downstream primer pairs, and enzyme-free water to 25 μL for PCR amplification. The reaction procedure was as follows: initial denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, and 63.8°C for 30 s, and extension at 72°C for 1 min. Amplified products were stored at 4°C. 5 μL of PCR product was analyzed by electrophoresis on a 1.5% agarose gel.

[0062] The multiplex PCR test at different temperatures is as follows Figure 10 As shown in the figure: annealing temperatures 1-7 are set to 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, and 63.8°C, respectively. Four clear bands can be seen at 63.8°C, indicating that the multiplex PCR method established in the present invention can simultaneously detect the minimum detection limit of neuronecrosis virus, iridescent virus, Streptococcus iniae, and Vibrio harveyi at 63.8°C, with high detection specificity and strong sensitivity.

[0063] Although specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these are merely examples and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention.

Claims

1. A multiplex PCR primer set for simultaneous detection of four pathogens, characterized in that: The four pathogens are neuronecrosis virus, iridovirus, Streptococcus iniae and Vibrio harveyi, and the primer set consists of SEQ ID NO. 1-8.

2. A multiplex PCR detection kit for simultaneous detection of four pathogens, characterized in that: The kit comprises the primer set according to claim 1.

3. A method for multiplex PCR detection using the primer set according to claim 1, characterized in that: The method is for the purpose of non-disease treatment and diagnosis. The system of the PCR detection method is as follows: 1 μL of the template of the sample to be tested, 12.5 μL of PCR mix, 1 μL of each primer in the primer set of claim 1, and enzyme-free water are added to 25 μL for PCR amplification; the reaction procedure is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 63.8°C for 30 seconds, extension at 72°C for 1 minute, amplification for 30 cycles, and storage of the amplified product at 4°C; the PCR product is subjected to agarose gel electrophoresis for detection; the target bands of neural necrosis virus, iridescent virus, Streptococcus iniae, and Vibrio harveyi are 544 bp, 472 bp, 325 bp, and 199 bp respectively.

4. The use of the primer set according to claim 1, characterized in that: The application is for non-disease diagnosis and treatment purposes, and the application method is to use the PCR primer set described in claim 1 to perform a PCR reaction, and perform agarose gel electrophoresis detection on the reaction products. The target bands of neural necrosis virus, iridescent virus, iniae Streptococcus and Vibrio harveyi are divided into 544bp, 472bp, 325bp and 199bp.

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