Primer probe combination and kit for on-site detection of fish viruses and bacteria

Abstract

The invention discloses a primer probe combination and a kit for on-site detection of fish viruses and bacteria, and relates to the technical field of gene detection. The invention provides a primer probe combination for detecting red sea bream iridovirus, nervous necrosis virus, vibrio harveyi and edwardsiella on site. The primer probe combination comprises a primer pair and a probe for detecting the red sea bream iridovirus, the primer pair and the probe are used for detecting the nervous necrosis virus; the primer pair and the probe are used for detecting vibrio harveyi; the invention discloses a primer pair and a probe for detecting edwardsiella. The method for detecting the fish viruses and bacteria by adopting the primer probe combination and the kit integrates sample pretreatment, nucleic acid extraction, purification, amplification and detection, has better sensitivity and accuracy, is simple and convenient to operate, is quick and time-saving, and is suitable for large-scale popularization and application. The method plays an important role in on-site rapid detection of research and safety management of fish viruses and bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, and in particular to a primer-probe combination and a kit for on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella. Background Art

[0002] Red seabream iridovirus (RSIV) belongs to the Iridoviridae family. It has an enveloped virus with an icosahedral nucleocapsid measuring 200-260 nm in diameter, and the viral nucleic acid is double-stranded linear DNA. The virus first broke out in Japanese red seabream farms in 1990, primarily affecting juvenile fish and resulting in a mortality rate of up to 37.9%. The disease peaks between July and October, with water temperatures between 22.6 and 25.5°C. Symptoms naturally subside when water temperatures drop below 18°C. It is a Category II infectious and parasitic disease. Red seabream iridovirus infection is primarily transmitted horizontally, spreading and infecting fish through the water, bait, and body.

[0003] Viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy, is a Class II infectious and parasitic disease. It is prevalent in marine fish species in nearly all regions except the Americas and Africa. It is highly harmful to larvae and young fish, with severe cases resulting in up to 100% mortality within a week. In recent years, the number of affected fish species and the extent of the disease have increased rapidly. Affected fish exhibit anorexia, float at the surface, swim in spirals or rotations, or float with their abdomens facing upward, making it difficult to sink. The abdomen is swollen, and some fish may have enlarged and congested swim bladders. No other obvious external changes are observed. Histological examination reveals vacuolation of central nervous system brain cells and retinal cells. The pathogen of this disease belongs to the Nodaviridae family.

[0004] Vibrio harveyi, also known as Vibrio harveyi, is a Gram-negative bacterium measuring 0.6–0.8 μm x 1.4–1.6 μm. It is a short, rod-shaped bacterium with blunt ends and a single polar flagellum approximately 3.6–4.5 μm long. It is highly contagious in species such as turbot, blue grouper, coioidal grouper, sea bass, large yellow croaker, and semi-smooth tongue sole.

[0005] Edwardsiella has a wide host range, infecting farmed mullet, sea bream, mullet, and yellowtail; freshwater eels, channel catfish, and tilapia can also be infected. Symptoms vary depending on the affected fish: Infected squid develop large ulcers on the abdomen and sides, bleeding at the edges and emitting a strong foul odor. The abdominal cavity fills with gas, causing the abdomen to swell. Infected mullet experience decreased food intake and a swollen abdomen. Dissections reveal egg-white-like ascites, enlarged kidneys, and numerous small white spots. Infected sea bream and red sea bream, the skin develops hemorrhagic ulcers, and numerous small white spots are found on the spleen and kidneys. Infections with these viruses or bacteria in aquaculture can significantly increase costs and cause inestimable losses to the industry.

[0006] Currently, there are also PCR detection methods for these viruses in the existing technology. Patent CN118086596A discloses a method for detecting neuronecrosis virus; Patent CN118480633A discloses a method for detecting red sea bream iridovirus and large yellow croaker iridovirus; Patent CN105296617B discloses a method for detecting Vibrio harveyi using a gene chip. The above methods are all for the detection of a single virus and lack the ability to detect two or more viruses simultaneously.

[0007] Based on the above problems, the inventors have developed for several years and disclosed a new on-site rapid nucleic acid detection equipment in the prior application CN202110057507.0. The equipment is a "handheld real-time fluorescence PCR all-in-one machine P1000F". After optimization and upgrading of temperature control, rapid nucleic acid detection can be completed within 35 minutes, and on-site automated detection of "sample in, result out" can be achieved. In order to solve the problem of sample pre-treatment, the inventors have developed a sample processing tube (ZL202320319535.X) that is easy to operate and fast. On the basis of sample processing and detection equipment, the present invention further develops an invention that can better cooperate with the equipment, namely, a primer probe combination and an integrated closed microfluidic chip kit and application for the joint detection of four bacterial viruses: red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella. The kit performs nucleic acid extraction, purification, amplification and detection of samples on an integrated chip, which is applied to rapid nucleic acid detection on site. Summary of the Invention

[0008] The purpose of the present invention is to provide a primer probe combination and a kit for on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella. The primer probe combination and the kit of the present invention are used to detect red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella, with good sensitivity and accuracy. The method is simple to operate, fast and time-saving.

[0009] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0010] On the one hand, the present invention provides a primer-probe combination for on-site detection of red sea bream iridovirus, nervous necrosis virus, Vibrio harveyi and Edwardsiella, the primer-probe combination comprising: primer pairs and probes for detecting red sea bream iridovirus, whose sequences are respectively shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting nervous necrosis virus, whose sequences are respectively shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting Vibrio harveyi, whose sequences are respectively shown in SEQ ID NOs: 7-9; primer pairs and probes for detecting Edwardsiella, whose sequences are respectively shown in SEQ ID NOs: 10-12; and primer pairs and probes for detecting IPC, whose sequences are respectively shown in SEQ ID NOs: 13-15.

[0011] Specifically, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a quencher group.

[0012] More specifically, the fluorescent reporter group is selected from one or more of FAM, ROX, HEX, CY5, VIC, TET, JOE, CY3, CY7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, ATTO 425, BODIPY FL, AlexaFluor488, YakimaYellow, Quasar 570, Aqua Phluor593, ATTO 590, and CY5.5.

[0013] More specifically, the fluorescent reporter group is selected from one or more of ATTO425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE, and ROX.

[0014] More specifically, the quencher group is a fluorescence quencher group.

[0015] More specifically, the fluorescence quenching group is selected from one or more of 6-TAMRA, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, QYS-7, and SQ1.

[0016] More specifically, the fluorescence quenching group is selected from one or more of BHQ2, BHQ3, SQ1, and MGB.

[0017] On the other hand, the present invention provides the use of the above primer-probe combination in the preparation of products for detecting red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella.

[0018] Specifically, the products include independent detection reagents, chips and test kits.

[0019] In another aspect, the present invention provides a kit for on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella, the kit comprising the above-mentioned primer-probe combination.

[0020] Specifically, the kit is an integrated closed microfluidic chip kit.

[0021] More specifically, the integrated closed microfluidic chip kit includes purification reagents, air-dried qPCR reagents and freeze-dried IPC quality control products.

[0022] Specifically, the purified reagents, air-dried qPCR reagents and freeze-dried IPC quality control products can be transported and stored at room temperature.

[0023] More specifically, the purification reagent includes a lysis solution, a washing solution, an eluent and magnetic beads, the lysis solution and the washing solution include guanidine hydrochloride, sodium acetate, Triton X-100 and 1,3-butanediol, the eluent includes Tris-HCl, and the magnetic beads are dried magnetic beads.

[0024] More specifically, the lysis solution and washing solution include 4.2M guanidine hydrochloride, 0.28M sodium acetate (pH 4.7), 1.4% Triton X-100 and 30% 1,3-butanediol, and the eluent includes 10mM Tris-HCl (pH 8.5).

[0025] Specifically, the kit completes nucleic acid extraction, purification, amplification and detection of samples on an integrated chip.

[0026] Specifically, the microfluidic chip kit can be used for the P1000F rapid nucleic acid detection device.

[0027] In another aspect, the present invention provides a method for on-site detection of red sea bream iridescent virus, neuronecrosis virus, Vibrio harveyi and Edwardsiella spp., the method comprising preparation of an integrated closed microfluidic chip kit, sample pre-treatment, and rapid device detection;

[0028] Specifically, the method comprises the following steps:

[0029] (1) preparing the above primer-probe combination into an integrated closed microfluidic kit;

[0030] (2) adding samples infected with red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella into a sample processing tube for processing to obtain a liquid to be tested;

[0031] (3) dropping the liquid to be tested into the integrated closed microfluidic reagent kit;

[0032] (4) Place the test kit containing the liquid to be tested on the testing equipment for testing and analysis.

[0033] Specifically, the step (2) is as follows: adding a sample pre-treatment solution and abrasive particles into a sample processing tube; the sample pre-treatment solution includes Tris-HCl (pH 8.0), NaCl, EDTA and SDS;

[0034] More specifically, the concentration range of Tris-HCl is 10mM-100mM, the concentration range of NaCl is 30mM-1.4M, the concentration range of EDTA is 1.5mM-20mM, and the concentration range of SDS is 0.05%-2%; the diameter of the grinding particles is 0.1 micron-1 mm.

[0035] More specifically, the concentration of the sample pretreatment solution Tris-HCl (pH 8.0) ranges from 30 mM to 55 mM, the concentration of NaCl ranges from 350 mM to 700 mM, the concentration of EDTA ranges from 5 mM to 10 mM, and the concentration of SDS ranges from 0.5% to 1%.

[0036] Specifically, the sample in step (2) is fish tissue infected with red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella.

[0037] Specifically, the step (4) includes:

[0038] 1) Turn on the rapid detection device;

[0039] 2) Scan the QR code;

[0040] 3) Insert the integrated microfluidic chip kit;

[0041] 4) Run the test;

[0042] 5) End and check the test results and amplification curve.

[0043] According to some embodiments of the present invention, the method comprises the following steps:

[0044] (1) preparing an integrated closed microfluidic kit by combining the above primers and probes;

[0045] (2) Rapid sample pre-processing using a sample processing tube;

[0046] (3) Using purification reagents on the chip to lyse the sample and extract and purify nucleic acids;

[0047] (4) using the primer-probe combination to rapidly amplify the sample nucleic acid extracted and purified in step (3);

[0048] (5) Analyze the amplification results.

[0049] In another aspect, the present invention provides use of the primer-probe combination, the kit, or the method in detecting fish infected with red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella.

[0050] The beneficial effects of the present invention are:

[0051] The present invention provides a primer-probe combination, an integrated microfluidic chip kit, and a method for detecting red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella. The method for detecting red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella using the primer-probe combination and kit of the present invention integrates sample pretreatment, nucleic acid extraction, purification, amplification, and detection, has good sensitivity and accuracy, and is simple to operate, fast, and time-saving. The method provided by the present invention can simultaneously detect four pathogens, red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella. A single kit can simultaneously detect RNA viruses, DNA viruses, and bacteria, and plays an important role in the on-site rapid detection of bacterial and viral infections in aquaculture fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the sensitivity test result of red sea bream iridovirus.

[0053] Figure 2 This is a graph showing the sensitivity test results for neuronecrosis virus.

[0054] Figure 3 This is the sensitivity test result of Vibrio harveyi.

[0055] Figure 4 This is the sensitivity test result of Edwardsiella.

[0056] Figure 5 This is a physical picture of the integrated microfluidic chip and sample processing tube.

[0057] Figure 6 This is the test result of actual fish tissue samples infected with red sea bream iridovirus.

[0058] Figure 7 This is the test result of actual fish tissue samples infected with neuronecrosis virus.

[0059] Figure 8 This is a diagram showing the test results of actual fish tissue samples infected with Vibrio harveyi.

[0060] Figure 9 This is a diagram showing the test results of actual fish tissue samples infected with Edwardsiella.

[0061] Figure 10 This is the result of the red sea bream iridovirus detection in comparative example 1.

[0062] Figure 11 This is a graph showing the results of neuronecrosis virus detection in comparative example 1.

[0063] Figure 12 This is a graph showing the detection results of Vibrio harveyi in Comparative Example 1.

[0064] Figure 13 This is the test result diagram of Edwardsiella sample in comparative example 1. DETAILED DESCRIPTION

[0065] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0066] The microfluidic chip device for nucleic acid detection of the present invention is detailed in patent 202110055537.8, the magnetic bead drying method is detailed in patent 202110222434.6, the sample processing tube is detailed in patent 202320319535.X, and the overall device, i.e., the PCR reaction device, is detailed in patent 202110057507.0. The actual picture of the integrated microfluidic chip and sample processing tube is as follows Figure 5 shown.

[0067] The fish tissues infected with red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella in the following examples were provided by the cooperative unit Yantai University.

[0068] Example 1 Primer probe combination for rapid on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella

[0069] Primer and probe sequences were designed based on the specific sequences of red sea bream iridovirus, neuronecrosis virus, Vibrio harveyi and Edwardsiella.

[0070] Among them, the primer and probe sequences of red sea bream iridovirus are:

[0071] RSIV-F: 5'-CGTAGTTCCTGGTACAGCAAG-3' (SEQ ID NO: 1);

[0072] RSIV-R: 5'-CTCCTGGCCGAAATTAGCAT-3' (SEQ ID NO: 2);

[0073] RSIV-P: 5'-CCCGTCACCCTATCAAAACAGACT-3' (SEQ ID NO: 3).

[0074] The primer and probe sequences for neuronecrosis virus are:

[0075] VNN-F:5'-AAAGCAGAACAGTCCGACCC-3'(SEQ ID NO:4);

[0076] VNN-R:5'-CACACAGGAGTATCAGCCGACCAG-3' (SEQ ID NO:5);

[0077] VNN-P: 5'-ACGCTGCTCCTTTCCCGACGA-3' (SEQ ID NO: 6).

[0078] The primer and probe sequences for Vibrio harveyi are:

[0079] Harvi-F:5'-CGGTTACCAACTGATTTGTTC-3' (SEQ ID NO:7);

[0080] Harvi-R:5'-ATTTTCTTCCTCTTGAGCATC-3' (SEQ ID NO:8);

[0081] Harvi-P: 5'-ATAGTGGGCTTAAACGTTCAACT-3' (SEQ ID NO: 9).

[0082] The primer and probe sequences for Edwardsiella are:

[0083] Edw-F: 5'-ACATGTTGAACTCGGCACG-3' (SEQ ID NO: 10);

[0084] Edw-R: 5'-GCTAAGACAACAGAGGACGGAT-3' (SEQ ID NO: 11);

[0085] Edw-P: 5'-CAGAGATGGCCAGACGCACCC-3' (SEQ ID NO: 12).

[0086] The primer and probe sequences for the internal process control (IPC) are:

[0087] IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO: 13);

[0088] IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO: 14);

[0089] IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO: 15).

[0090] The primer-probe composition of red sea bream iridovirus, nervous necrosis virus, Vibrio harveyi and Edwardsiella of the present invention is used for detecting red sea bream iridovirus, nervous necrosis virus, Vibrio harveyi and Edwardsiella jointly or individually.

[0091] In the embodiment, the RSIV probe is labeled with ATTO 425, and the quenching group is MGB, the VNN probe is labeled with FAM, and the quenching group is SQ1, the Harvi probe is labeled with HEX, and the quenching group is BHQ2, the Edw probe is labeled with CY5, and the quenching group is BHQ2, and the IPC probe is labeled with CY5.5, and the quenching group is BHQ2.

[0092] Example 2 A microfluidic chip kit for detecting red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella

[0093] (1) The kit includes the primer-probe combination described in Example 1, with final concentrations as shown in Table 1 below.

[0094] Table 1 Primer and probe concentrations

[0095] Primer probe name Final concentration RSIV-F, RSIV-R 0.2μM, 0.2μM RSIV-P 0.2μM VNN-F, VNN-R 0.2μM, 0.2μM VNN-P 0.2μM Harvi-F, Harvi-R 0.2μM, 0.2μM Harvi-P 0.2μM Edw-F, Edw-R 0.2μM, 0.2μM Edw-P 0.2μM IPC-F, IPC-R 0.2μM, 0.2μM IPC-P 0.2μM

[0096] (2) The kit also includes purification reagents and air-dried reagents.

[0097] 1) The purification reagent includes 450 μL of lysis solution, 450 μL of washing solution and 850 μL of eluent, and 15 μL of magnetic beads. The lysis solution and washing solution include 4.2 M guanidine hydrochloride, 0.28 M pH 4.7 sodium acetate, 1.4% Triton X-100 and 30% 1,3-butanediol. The eluent includes 10 mM pH 8.5 Tris-HCl. The purification reagent is added to the sample loading layer in the chip. The magnetic bead drying method is described in patent 202110222434.6.

[0098] 2) The 50 μL air-dried reagent system is shown in Table 2 below.

[0099] Table 2 Air-drying reagent system

[0100] Element volume <![CDATA[4×Air-Dryable TM Direct RNA / DNA qPCR Mix]]> 12.5μL Primer probe mixture 2.5 μL

[0101] Add the reagents in Table 2 above to the reaction chamber of the integrated microfluidic chip, place the chip in an air drying oven, and dry it according to the Air-Dryable TM Air-dry according to the Direct RNA / DNA qPCR Mix instructions.

[0102] 3) The freeze-dried IPC quality control product includes the preparation of IPC pseudovirus and a freeze-dried reagent system.

[0103] According to the pseudovirus preparation method described in Example 1 of patent CN202110669475.X, a pseudovirus of the IPC gene was constructed and obtained.

[0104] The concentration was 1×10 6 copies / mL IPC pseudovirus 5μL was prepared into lyophilized pellets using a common lyophilization system and method;

[0105] Table 3 Lyophilized IPC reagent system

[0106] Element content IPC pseudovirus <![CDATA[1×10 6 copies / mL]]> Mannitol 10％ Trehalose 10％ Pullulan 0.2％ Bovine serum albumin (BSA) 1mg / mL Defoaming agent SE-15 0.036％ Tris-HCl pH 8.0 10mM NaCl 50mM Tween20 0.05％

[0107] 5 μL of the reagents in Table 3 were dropped into liquid nitrogen to prepare freeze-dried pellets, which were then placed in vials and freeze-dried according to the freeze-drying program in Table 4 below.

[0108] Table 4 Freeze-drying procedure

[0109]

[0110] The freeze-dried balls are placed on the chip with air-dried reaction reagents to assemble an integrated detection kit.

[0111] Example 3 Positive sample preparation

[0112] The positive quality control products used in the test are all from the full-gene synthesis plasmid of Qingke Biotechnology. Detailed information is shown in Table 5 below.

[0113] Table 5 Target information

[0114] Target information copies / ng Length (bp) Vibrio harveyi (fish) 2.79E+08 3275 Edwardsiella (fish) 2.81E+08 3243 Neuronal necrosis virus (fish) 2.80E+08 3257 Red sea bream iridovirus (fish) 2.67E+08 3416

[0115] Example 4 Preparation of sample processing tube

[0116] (1) Install the sample processing tube;

[0117] (2) Add about 1 g of diamond abrasive to a tube and add 3 mL of sample pretreatment solution (50 mM Tris-HCl (pH 8.0), 700 mM NaCl, 10 mM EDTA, 1% SDS) to the tube;

[0118] (3) Place the tube in the tooling position of a precision manual press and wrap it in 160°C aluminum foil.

[0119] Example 5 Detection Methods for Red Sea Bream Iridovirus, Neurological Necrosis Virus, Vibrio harveyi and Edwardsiella

[0120] (1) Remove the aluminum foil protective film from the sample processing tube, place it upright, add the fish tissue to be tested into the sample processing tube, and tighten the tube cap;

[0121] (2) Keep the tube upright and rub it 5-10 times to ensure that the sample is fully in contact with the grinding material and that the grinding is sufficient;

[0122] (3) Open the outer packaging, take out the reagent chamber and reaction layer of the chip, tear off the sealing strip on the back of the reagent chamber, hold the reagent chamber and reaction layer so that the head of the reaction layer is vertically upward, press hard to puncture the reagent chamber until the upper and lower layers of the chip are seamlessly connected, and complete the assembly;

[0123] (4) Unscrew the sample chamber cover, add 2-3 drops (50 μL) of sample grinding solution into the sample chamber, and tighten the sample chamber cover;

[0124] (5) Press and hold the device power button for more than 3 seconds to turn on the device, and click the "Test" button on the homepage;

[0125] (6) After waiting for 3 seconds, you will see the red light flashing in the scanning window and scan the chip QR code;

[0126] (7) Scan the sample information or skip according to the on-screen prompts;

[0127] (8) Follow the on-screen instructions to open the hatch, insert the chip, and close the hatch;

[0128] (9) Click the "Run" button to start the test;

[0129] (10) After the operation is completed, check the test results.

[0130] Sample processing time in the sample handling tube is approximately 1 minute; nucleic acid extraction and purification on the integrated microfluidic chip takes approximately 12 minutes. The real-time quantitative PCR reaction protocol on the integrated microfluidic chip is: 95°C for 1 minute; (95°C for 5 seconds, 60°C for 8 seconds), 45 cycles, for a total amplification time of 24 minutes. The entire process for testing for red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella pneumoniae, from sample processing, nucleic acid extraction and purification, to amplification and test results, takes approximately 37 minutes.

[0131] Experimental Example 1 Sensitivity Detection

[0132] Samples containing 500, 250, and 100 copies of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella plasmid were prepared for nucleic acid amplification detection. The system components are shown in Table 2. The test results are shown in Table 6 below. The test curves are shown in Table 6 below. Figure 1-Figure 4 shown.

[0133] Table 6 Sensitivity test results

[0134]

[0135]

[0136] As can be seen from Table 6 above, the primer composition and kit described in the present application have good sensitivity and can detect plasmid samples of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella with a content of 100 copies.

[0137] Experimental Example 2: Actual Sample Testing

[0138] Fish meat (0.2 g) infected with different types of viruses were used in the experiments.

[0139] The above-extracted samples were tested according to the system and steps of Examples 1-5. The test results are shown in Table 7 below. Figure 6-Figure 9 shown.

[0140] Table 7 Actual sample test results

[0141] Virus type Target Ct value IPC internal reference Ct value Test results Red sea bream iridovirus 32.26 29.09 Positive Neuronecrosis virus 30.52 30.08 Positive Vibrio harveyi 30.51 29.91 Positive Edwardsiella 32.15 30.23 Positive

[0142] It can be seen from Table 7 that the primer-probe compositions for red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella spp. of the present invention can be used to detect real samples and can be used for on-site rapid identification.

[0143] Comparative Example 1

[0144] The difference between Comparative Example 1 and Examples 1-4 and Experimental Example 1 is only the difference in the primer and probe sequences, and all other aspects are the same. The primer and probe sequences of Comparative Example 1 are as follows:

[0145] Among them, the primer and probe sequences of red sea bream iridovirus are:

[0146] RSIV-F: 5'-CGGCCAGGAGTTTAGTGTGACT-3' (SEQ ID NO: 16);

[0147] RSIV-R: 5'-GCTGTTCTCCTTGCTGGACG-3' (SEQ ID NO: 17);

[0148] RSIV-P: 5'-TGTGGCTGCGTGTTAAGATCCCCTCCA-3' (SEQ ID NO: 18).

[0149] The primer and probe sequences for neuronecrosis virus are:

[0150] VNN-F:5'-CTTCCTGCCTGATCCAACTG-3'(SEQ ID NO:19);

[0151] VNN-R:5'-GTTCTGCTTTCCCACCATTTG-3' (SEQ ID NO:20);

[0152] VNN-P: 5'-CAACGACTGCACCACGAGTTG-3' (SEQ ID NO: 21).

[0153] The primer and probe sequences for Vibrio harveyi are:

[0154] Harvi-F:5'-CCACTGCTGAGACAAAAGCA-3' (SEQ ID NO:22);

[0155] Harvi-R:5'-GTGATTCTGCAGGGTTGGTT-3' (SEQ ID NO: 23);

[0156] Harvi-P: 5'-CAGCCGTCGAACAAGCACCG-3' (SEQ ID NO: 24).

[0157] The primer and probe sequences for Edwardsiella are:

[0158] Edw-F: 5'-CAGTGATAAAAAGGGTGGA-3' (SEQ ID NO: 25);

[0159] Edw-R: 5'-CTACACAGCAACGACAACG-3' (SEQ ID NO: 26);

[0160] Edw-P: 5'-AGACAACAGAGGACCGGATGTGGC-3' (SEQ ID NO: 27).

[0161] The real samples of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella were put into the test. The experimental results of Experimental Example 2 and Comparative Example 1 are shown in Table 8 below. Figure 10-13 shown.

[0162] Table 8 Actual sample test results

[0163] Virus type Experimental Example 2 Ct value Comparative Example 1 Ct value Red sea bream iridovirus 32.26 39.50 Neuronecrosis virus 30.52 38.05 Vibrio harveyi 30.51 36.43 Edwardsiella 32.15 (-)

[0164] As can be seen from the figure, the fluorescence values ​​of the comparative examples are generally low, and some Ct values ​​are also delayed, indicating that their detection sensitivity is low.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer-probe combination for on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella, characterized in that: The primer-probe combination includes: primer pairs and probes for detecting the sequences of red sea bream iridovirus as shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting the sequences of neural necrosis virus as shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting the sequences of Vibrio harveyi as shown in SEQ ID NOs: 7-9; primer pairs and probes for detecting the sequences of Edwardsiella as shown in SEQ ID NOs: 10-12; and primer pairs and probes for detecting the sequences of IPC as shown in SEQ ID NOs: 13-15.

2. Use of the primer-probe combination according to claim 1 in the preparation of a product for detecting red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella.

3. The use according to claim 2, characterized in that The products include independent detection reagents, chips and test kits.

4. A kit for on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella, characterized in that: The kit comprises the primer-probe combination according to claim 1.

5. The kit according to claim 4, characterized in that The test kit is an integrated closed microfluidic chip test kit.

6. The kit according to claim 5, characterized in that The integrated closed microfluidic chip kit includes purification reagents, air-dried qPCR reagents and freeze-dried IPC quality control products.

7. A method for on-site detection of red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella, characterized in that: The method comprises the following steps: (1) preparing the primer-probe combination described in claim 1 into an integrated closed microfluidic kit; (2) adding a sample containing red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella into a sample processing tube for processing to obtain a liquid to be tested; (3) dropping the liquid to be tested into the integrated closed microfluidic reagent kit; (4) Place the test kit containing the liquid to be tested on the testing equipment for testing and analysis.

8. The method according to claim 7, characterized in that The step (2) specifically comprises: adding a sample pre-treatment solution and abrasive particles into the sample processing tube; The sample pretreatment solution includes Tris-HCl (pH 8.0), NaCl, EDTA and SDS; The sample described in step (2) is fish infected with red sea bream iridovirus, neural necrosis virus, Vibrio harveyi and Edwardsiella.

9. The method according to claim 7, characterized in that The step (4) specifically includes: 1) Turn on the rapid detection device; 2) Scan the QR code; 3) Inserting the integrated microfluidic chip kit; 4) Run the test; 5) End and check the test results and amplification curve.

10. Use of the primer-probe combination according to claim 1, the kit according to any one of claims 4 to 6, or the method according to any one of claims 7 to 9 in detecting fish infected with red sea bream iridovirus, neural necrosis virus, Vibrio harveyi, and Edwardsiella.

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