Multiplex PCR (Polymerase Chain Reaction) primer group for simultaneously detecting four pathogens, detection method and application

Abstract

The invention relates to a multiplex PCR (polymerase chain reaction) primer group for simultaneously detecting four pathogens, a detection method and application, and belongs to the field of molecular biology, the four pathogens are nervous necrosis virus, iridovirus, streptococcus iniae and vibrio harveyi, and the primer group is composed of SEQ ID NO.1-8. By utilizing the primer, nervous necrosis virus, iridovirus, streptococcus iniae and vibrio harveyi can be simultaneously detected in a short time, and the aim of establishing a simple and effective method for simultaneously detecting various fish pathogens is fulfilled. And the detection primer group and the detection method provided by the invention have good specificity and relatively 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 thereof. Background Art

[0002] Nervous Necrosis Virus (NNV) belongs to the family Nodaviridae and the genus Betanodavirus. This genus of viruses specifically infects fish and can cause viral neuronecrosis in fish. NNV consists of two single-stranded positive RNAs, including one RNA1 that encodes RNA polymerase and one RNA2 that encodes its unique capsid protein in different species. NNV can be divided into five types based on the homology of its RNA2, namely red-spotted grouper neural necrosis virus (RGNNV), turbot neural necrosis virus (TNNV), redfin puffer neural necrosis virus (TPNNV), yellow-banded trevally neural necrosis virus (SJNNV) and striped flounder neural necrosis virus (BFNNV). In the early detection of NNV, most of them use the unique characteristics of RNA2 in different virus species to design primers for RNA2 single-stranded sequences.

[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 endangers the survival of fish. RSIV is latent and has a high mortality rate, making it difficult to prevent and control clinically.

[0004] Streptococcus iniae belongs to the order Lactobacilales, the family Streptococcus, the genus Streptococcus, and is a Gram-positive bacterium. It is one of the important pathogens of aquatic animals and has a wide range of host adaptability. The host range includes dozens of freshwater fish such as tilapia, rainbow trout, turbot, and ovalbumin, becoming one of the main pathogens of fish invasive infections and outbreaks worldwide. The treatment of streptococcal disease in dolphins mainly relies on the use of multiple antibiotics. The use of a large number of antibiotics not only leads to bacterial resistance, but also destroys the stability of water bodies. Therefore, early prevention of streptococcus iniae is particularly important.

[0005] Vibrio harveyi is a major pathogenic vibrio of many aquatic animals, widely distributed in aquaculture seawater and the body and outside of aquatic animals. In recent years, frequent outbreaks of vibriosis caused by Vibrio harveyi have seriously affected the development of my country's aquaculture industry. Studies have shown that the reported Vibrio harveyi can infect important aquatic economic animals such as fish, shrimp and shellfish such as sea bream, white shrimp, sea bass and abalone.

[0006] Traditional methods for identifying bacteria and viruses are cumbersome, complex, and time-consuming. In recent years, immunology and molecular biology techniques have continued to advance, such as real-time fluorescence quantitative PCR, LAMP, and enzyme-linked immunosorbent assay. These techniques have certain limitations in detection time and sensitivity. In contrast, PCR technology has high sensitivity, strong specificity, and is easy to operate. However, there are few studies on the simultaneous detection of neural necrosis virus, iridovirus, Streptococcus dolphinus, and Vibrio harveyi. Therefore, a multiplex PCR detection method that can simultaneously detect neural necrosis virus, iridovirus, Streptococcus dolphinus, and Vibrio harveyi has important practical significance and application value. Summary of the invention

[0007] The purpose of the present invention is to provide a primer set and a detection method that can simultaneously detect neural necrosis virus, iridovirus, Streptococcus iniae and Vibrio harveyi in a short time, so as to achieve the purpose of establishing a simple and effective method for simultaneously detecting multiple fish pathogens. The technical problem to be solved by the present invention is to provide a multiplex PCR system for simultaneously detecting neural necrosis virus, iridovirus, Streptococcus iniae and Vibrio harveyi, and the amplified products can be amplified and detected using agarose gel electrophoresis technology.

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

[0009] A multiplex PCR detection kit for simultaneously detecting four pathogens, wherein the kit comprises the primer set.

[0010] The present invention also provides a method for multiplex PCR detection using the primer set, wherein 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; reaction procedure: pre-denaturation at 95° C. for 5 min; denaturation at 95° C. for 30 s, annealing at 63.8° C. for 30 s, extension at 72° C. for 1 min, amplification for 30 cycles, and storage of the amplified products at 4° C.; taking the PCR products for detection by agarose gel electrophoresis, and the target bands of the neural necrosis virus, iridovirus, Streptococcus dolphinus and Vibrio harveyi are 544 bp, 472 bp, 325 bp and 199 bp respectively.

[0011] The present invention also provides an application of the primer set, the application is for non-disease detection and treatment purposes, and the application method is to use the PCR primer set to perform a PCR reaction, and perform agarose gel electrophoresis on the reaction product. The target bands of neural necrosis virus, iridovirus, Streptococcus dolphinus and Vibrio harveyi are divided into 544bp, 472bp, 325bp and 199bp.

[0012] The beneficial effects of the present invention compared with the prior art: The detection sensitivity and specificity of the multiple PCR amplification system of the present invention were tested, and the sensitivity test results showed that the sensitivity of using PCR to detect Streptococcus dolphins, Vibrio harveyi, neuronecrosis virus, and iridovirus has a wide detection range, and can be detected in samples within a certain range. The specific detection results show that the method is used to detect Streptococcus dolphins, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio cannelli, Staphylococcus aureus, Vibrio parahaemolyticus, Vibrio rotifer, Staphylococcus epidermidis, Lactococcus gasseri, Photobacterium mermaids, and Streptococcus dysgalactiae. As a result, only Streptococcus dolphins, Vibrio harveyi, neuronecrosis virus, and iridovirus can be amplified well, while other bacteria cannot be amplified. Therefore, it is shown that the primer set has good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the specificity test results of the neural necrosis virus primers; M: Marker, 1: Streptococcus iniae DNA, 2: Vibrio harveyi DNA, 3: neural necrosis virus cDNA, 4: iridovirus DNA, 5: Lactococcus garvieae DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannelli DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control; Figure 2 Schematic diagram of the specificity test results of iridovirus primers; M: Marker, 1: Streptococcus dolphinus DNA, 2: Vibrio harveyi DNA, 3: Neuronecrosis virus cDNA, 4: iridovirus DNA, 5: Lactococcus garrisonii DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannelli DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control; Figure 3 Schematic diagram of the results of 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 garvei DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannelli DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control; Figure 4 Schematic diagram of the results of primer specificity test for Vibrio harveyi; M: Marker, 1: Streptococcus dolphinus DNA, 2: Vibrio harveyi DNA, 3: Neuronecrosis virus cDNA, 4: Iridovirus DNA, 5: Lactococcus garrisonii DNA, 6: Staphylococcus epidermidis DNA, 7: Staphylococcus aureus DNA, 8: Streptococcus dysgalactiae DNA, 9: Vibrio parahaemolyticus DNA, 10: Vibrio cannelli DNA, 11: Vibrio rotifer DNA, 12: Photobacterium mermanii DNA, N: negative control; Figure 5 Schematic diagram of the specificity test results of the primers of neural necrosis virus, iris virus, Streptococcus iniae and Vibrio harveyi in a multiplex system; M: Marker, 1: neural necrosis virus cDNA, 2: iris virus DNA, 3: Streptococcus iniae DNA, 4: Vibrio harveyi DNA, 5: mixed DNA of neural necrosis virus, iris virus, Streptococcus iniae and Vibrio harveyi, 6: negative control of lane 1, 7: negative control of lane 2, 8: negative control of lane 3, 9: negative control of lane 4, 10: negative control of lane 5; 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; Figure 7Schematic diagram of the results of the iridovirus sensitivity test; M: Marker, 1: 20.8 ng / μL, 2: 2.08 ng / μL, 3: 208 pg / μL, 4: negative control; 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; Fig. 9 Schematic diagram of the results of the Vibrio harveyi sensitivity test; M: Marker, 1: 45.6 ng / μL, 2: 4.56 ng / μL, 3: 456 pg / μL, 4: negative control; Fig.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

[0014] The technical solution of the present invention is further explained below by way of embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.

[0015] Embodiment 1: (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'.

[0016] (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 oligo. The designed primer sequences are as follows: RSIV-MCP-F: 5'-CCGCCGCCTACCTTAATTTGCC-3'; RSIV-MCP-R: 5'-TTGTTGTTGACATACACGGGAC-3'.

[0017] (3) Design of PCR primers targeting the simA gene of Streptococcus india: 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', simA-R:5'-ACTTGCTGTGAAGAAAGGGTTA-3'.

[0018] (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), primers were designed using oligo. The designed primer sequences are as follows: toxR-F: 5'-ACCTAGAGCAATTCGCAGAG-3', toxR-R:5'-TGACGAAATTGTGATTCTGC-3'.

[0019] The above primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0020] Example 2 Extraction of DNA and cDNA of Streptococcus dolphins, Vibrio harveyi, neuronecrosis virus, and iridovirus: For DNA extraction of Streptococcus dolphins and Vibrio harveyi, 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.

[0021] The above-mentioned NNV-RNA2 primers were used to specifically detect 12 marine pathogenic bacteria (Table 1) at the same time, and the specificity of the amplification primers was analyzed and evaluated: Table 1 Strains used for PCR primer specificity analysis .

[0022] DNA or cDNA of 12 bacteria and viruses including Streptococcus dolphinus, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campestris, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garris, Vibrio rotiferus, Photobacterium mermaidii, and Streptococcus dysgalactiae were used as templates, and NNV-RNA2 of neuronecrosis virus was used as primers to construct a 20 μL reaction system. Each system group included 1 μL of 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 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, amplification for 30 cycles, and the amplified product was stored at 4°C. 5 μL of PCR product was taken for detection by 1.5% agarose gel electrophoresis. The results of the specificity verification of the amplification primers 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.

[0023] The RSIC-MCP primers of the above-mentioned iridovirus were used to specifically detect 12 marine pathogenic bacteria (Table 1) at the same time, and the specificity of the amplification primers was analyzed and evaluated: DNA or cDNA of 12 bacteria and viruses such as Streptococcus dolphinus, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campbellii, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garvieae, Vibrio rotiferus, Photobacterium mermaidii, and Streptococcus dysgalactiae were used as templates, and RSIV-MCP of iridovirus was used as primers to construct a 20μL reaction system. Each group of systems included 1μL of the upper and lower primers, 10μL of mix, 1μL of template, 7μL of DEPC water, and pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 30s, annealing at 64℃ for 30s, extension at 72℃ for 1min, amplification for 30 cycles, and the amplified products were stored at 4℃. Take 5μL of PCR products and perform electrophoresis on 1.5% agarose gel for detection. The specificity verification results of 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.

[0024] The sima primers of Streptococcus dolphins were used to specifically detect 12 marine pathogenic bacteria (Table 1) at the same time, and the specificity of the amplification primers was analyzed and evaluated: DNA or cDNA of 12 bacteria and viruses such as Streptococcus dolphins, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio campbellii, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garris, Vibrio rotiferus, Photobacterium mermaids, and Streptococcus dysgalactiae were used as templates, and sima of Streptococcus dolphins was used as primers to construct a 20μL reaction system. Each group of systems included 1μL of the upper and lower primers, 10μL of mix, 1μL of template, 7μL of DEPC water, and pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 30s, annealing at 57℃ for 30s, extension at 72℃ for 1min, amplification for 30 cycles, and the amplification products were stored at 4℃. Take 5μL of PCR product and perform electrophoresis on 1.5% agarose gel for detection. The specificity verification results of the amplification primers are shown in the figure. Figure 3 As shown, except for Streptococcus iniae which had an amplification band and was positive, the results of the other 11 pathogenic bacteria and the negative control were all negative, indicating that the simA amplification primers of the present invention have good specificity.

[0025] The toxR primers of Vibrio harveyi were used to specifically detect 12 marine pathogenic bacteria (Table 1) at the same time, and the specificity of the amplification primers was analyzed and evaluated: DNA or cDNA of 11 bacteria and viruses such as Streptococcus suis, Vibrio harveyi, neuronecrosis virus, iridovirus, Vibrio cannelli, Vibrio parahaemolyticus, Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus garveyi, Vibrio rotiferus, Photobacterium mermaids, and Streptococcus dysgalactiae were used as templates, and toxR of Vibrio harveyi was used as primers to construct a 20μL reaction system. Each group of systems included 1μL of the upper and lower primers, 10μL of mix, 1μL of template, 7μL of DEPC water, and pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 30s, annealing at 59℃ for 30s, extension at 72℃ for 1min, amplification for 30 cycles, and the amplified products were stored at 4℃. Take 5μL of PCR products and perform electrophoresis on 1.5% agarose gel for detection. The specificity verification results of amplification primers are as follows Figure 4 As shown, except for Vibrio harveyi which had an amplification band and was positive, the results of the other 11 pathogenic bacteria and the negative control were all negative, indicating that the toxR amplification primers of the present invention have good specificity.

[0026] DNA or cDNA of Streptococcus dolphinus, Vibrio harveyi, neuronecrosis virus, and iris virus were selected as templates, and amplification detection was performed using simA, toxR, NNV-RNA2, and RSIV-MCP primers. The results are as follows Figure 5It was shown that Streptococcus iniae, Vibrio harveyi, neuronecrosis virus and iridovirus were amplified and detected. Therefore, it was shown that the four pairs of primers were highly specific and could be used for simultaneous detection of neuronecrosis virus, iridovirus, Streptococcus iniae and Vibrio harveyi in a multiplex PCR reaction system.

[0027] Example 3 This example is about establishing sensitivity: a neural necrosis virus with an initial concentration of 17.4 ng / μL was selected, diluted in a ten-fold gradient, and the single sensitivity was measured. 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.

[0028] Establish a PCR reaction system, 1μL of cDNA at each concentration of neuronecrosis virus, 10μL of PCR mix, 1μL of upstream and downstream primers, and 20μL of enzyme-free water for PCR amplification. Reaction procedure: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 1min, amplification for 30 cycles, and the amplified product is stored at 4℃. Take 5μL of PCR product for detection by 1.5% agarose gel electrophoresis.

[0029] Sensitivity test of neuronecrosis virus Figure 6 As shown, the minimum detection limit was 1.74 ng / μL.

[0030] 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.

[0031] Establish a PCR reaction system, 1μL of DNA at each concentration of iridovirus, 10μL of PCR mix, 1μL of upstream and downstream primers, and 20μL of enzyme-free water for PCR amplification. Reaction procedure: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 64℃ annealing for 30s, 72℃ extension for 1min, amplification for 30 cycles, and the amplified product is stored at 4℃. Take 5μL of PCR product for detection by 1.5% agarose gel electrophoresis.

[0032] Sensitivity test for iridovirus Figure 7 As shown, the minimum detection limit was 2.08 ng / μL.

[0033] The initial concentration of Streptococcus dolphins was 42.1 ng / μL, and it was diluted in a ten-fold gradient to measure the single sensitivity. The concentrations of Streptococcus dolphins DNA were 42.1 ng / μL, 4.21 ng / μL, and 421 pg / μL, respectively.

[0034] Establish a PCR reaction system, 1μL of DNA of each concentration of S. iniae, 10μL of PCR mix, 1μL of upstream and downstream primers, and 20μL of enzyme-free water for PCR amplification. Reaction procedure: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 1min, amplification for 30 cycles, and the amplified product is stored at 4℃. Take 5μL of PCR product for detection by 1.5% agarose gel electrophoresis.

[0035] Susceptibility test for Streptococcus iniae Figure 6 As shown, the minimum detection limit was 4.21 ng / μL.

[0036] An initial concentration of 45.6 ng / μL of Vibrio harveyi was selected and diluted in a ten-fold gradient to measure the single sensitivity. The concentrations of DNA of Vibrio harveyi were 45.6 ng / μL, 4.56 ng / μL, and 456 pg / μL, respectively.

[0037] Establish a PCR reaction system, 1μL DNA of each concentration of Vibrio harveyi, 10μL PCR mix, 1μL upstream and downstream primers, 20μL enzyme-free water, and perform PCR amplification. Reaction procedure: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 59℃ annealing for 30s, 72℃ extension for 1min, amplification for 30 cycles, and the amplified product is stored at 4℃. Take 5μL PCR product and perform detection on 1.5% agarose gel electrophoresis.

[0038] Susceptibility testing of Vibrio harveyi Figure 7 As shown, the minimum detection limit was 4.56 ng / μL.

[0039] The minimum detection limits of Streptococcus iniae, Vibrio harveyi, neuronecrosis virus and iridovirus were tested at different temperatures in a multiplex PCR system.

[0040] The minimum detection limit of an equal mixture of Streptococcus iniae, Vibrio harveyi, neuronecrosis virus, and iridovirus.

[0041] Establish a multiplex PCR reaction system: 4 kinds of DNA and cDNA mixed templates, PCR mix 12.5μL, 1μL of each of the four pairs of upstream and downstream primers mentioned above, and add enzyme-free water to 25μL for PCR amplification. Reaction procedure: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, annealing at 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 63.8℃ for 30s, extension at 72℃ for 1min, amplification for 30 cycles, and the amplified products are stored at 4℃. Take 5μL of PCR products for detection by 1.5% agarose gel electrophoresis.

[0042] The multiplex PCR test at different temperatures is as follows Fig.10 As shown in the figure: the annealing temperatures 1-7 are set to 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, and 63.8°C, respectively, and 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, iridovirus, Streptococcus dolphinus and Vibrio harveyi at 63.8°C, and the detection has high specificity and strong sensitivity.

[0043] Although specific embodiments of the present invention are described above, those skilled in the art should understand that these are merely examples and that various changes or modifications may be made to these embodiments without violating the principles and essence of the present invention.

Claims

1. A multiplex PCR primer set for simultaneously detecting 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 detecting four pathogens simultaneously, characterized in that: The kit comprises the primer set according to claim 1.

3. A method for multiplex PCR detection using the primer set of claim 1, characterized in that: 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 to make up to 25 μL, and PCR amplification; reaction procedure: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 63.8°C annealing for 30 seconds, 72°C extension for 1 minute, amplification for 30 cycles, and the amplified product is stored at 4°C; the PCR product is taken for detection by agarose gel electrophoresis; the target bands of neural necrosis virus, iridovirus, iniae streptococcus and Vibrio harveyi are 544 bp, 472 bp, 325 bp and 199 bp.

4. The use of the primer set according to claim 1, characterized in that: The application is for non-disease detection 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 product. The target bands of neural necrosis virus, iridovirus, Streptococcus dolphinus and Vibrio harveyi are 544bp, 472bp, 325bp and 199bp.

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