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

Abstract

The invention discloses a primer probe combination and a kit for on-site detection of fish viruses, and relates to the technical field of gene detection. The invention provides a primer probe combination for on-site detection of a koi herpesvirus, a carp spring virus, a carp edema virus and a goldfish hematopoietic necrosis virus. The primer probe combination comprises a primer pair and a probe for detecting the koi herpesvirus, the primer pair and the probe are used for detecting the spring carp virus; the primer pair and the probe are used for detecting the carp edema virus; the invention relates to a primer pair and a probe for detecting goldfish hematopoietic necrosis virus. The method for detecting the fish viruses by adopting the primer probe combination and the kit disclosed by the invention integrates sample pretreatment, nucleic acid extraction, purification, amplification and detection, has better sensitivity and accuracy, is simple, convenient, rapid and time-saving to operate, and plays an important role in on-site rapid detection of research and safety management of the fish viruses.

Description

Technical Field

[0001] The present 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 koi herpesvirus, carp spring virus, carp edema virus and goldfish hematopoietic necrosis virus. Background Art

[0002] Koi herpesvirus infection (KHV) is a disease of koi carp and common carp caused by the koi herpesvirus (KHV). Affected fish exhibit sluggish reactions, loss of appetite, difficulty breathing, and ataxia; their gills may be hemorrhagic and necrotic, their eyes sunken, their skin may develop grayish-white spots, and their mucus secretions may be increased. KHV, also known as Cyprinid herpesvirus type 3 (CyHV-3), belongs to the Herpesviridae family and the Cyprinid herpesvirus genus. It is also known as Cyprinid herpesvirus type 3 (CyHV-3), a member of the same genus as carp pox virus (Cyprinid herpesvirus type 1, CHV-1) and goldfish hematopoietic necrosis virus (Cyprinid herpesvirus type 2, CyHV-2). While KHV shares morphological characteristics with the other two fish herpesviruses, it differs from them in clinical symptoms, host range, antigenicity, growth characteristics, and cytopathic patterns.

[0003] Spring viraemia of carp (SVCV) is an acute, hemorrhagic, infectious disease of fish caused by the Spring viraemia of carp virus (SVCV). It primarily infects common carp and is characterized by systemic bleeding and ascites, a rapidly progressive condition, and a high mortality rate. Transmission occurs through water, where the virus can enter fish through the gills and intestines. It can also be transmitted vertically or mechanically by certain aquatic blood-sucking parasites. The virus is found in the urine and secretions of infected and infected fish. Common reservoirs include common carp, goldfish, and crucian carp.

[0004] Carp edema disease, also known as koi sleeping sickness, is a serious acute infectious disease of common carp and ornamental koi caused by the carp edema virus (CEV). Mortality rates can reach 80-100%. Affected fish become lethargic, gather below the water's surface, or lie on the bottom of the pond, ultimately dying within two weeks. Symptoms include edema, retracted eyes, swollen and hypertrophic gills, congested tail fins, and red and swollen anus. Furthermore, mucus secreted by the fish easily rubs off, revealing rough scales. Dissection reveals kidney erosion and brittle intestinal texture.

[0005] Goldfish hematopoietic necrosis (GFHN) is a new aquatic animal disease caused by carp herpesvirus type II infecting crucian carp or goldfish. It is classified as a Class II aquatic animal disease in the 2022 "List of Class I, II and III Animal Diseases". The prevalence of this disease in recent years has caused serious economic losses to the crucian carp and goldfish farming industries. The herpesvirus has a unique latent-reactivation mechanism, which can shut down most active genes and only turn on a few specific genes in a specific latent stage. Under appropriate conditions, it resumes activity and re-enters the proliferation process. This "latent-relapse" and "multi-pathogen mixed infection" epidemic characteristics have brought huge challenges to the analysis of pathogen transmission patterns and virus prevention and control.

[0006] Currently, existing methods exist for detecting these viruses. For example, patent CN116656879A discloses a method for simultaneously detecting carp herpesvirus, carp spring virus, and carp edema virus using loop-mediated isothermal amplification; patent CN118064645A discloses a method for detecting goldfish hematopoietic necrosis virus; and patent CN111635961B discloses a method for detecting koi herpesvirus. These methods can detect several viruses individually or in combination, but they are time-consuming and require specialized laboratories and personnel, making them less suitable for on-site testing.

[0007] Based on the above problems, the inventors have developed for several years and disclosed a new type of 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 koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic organ necrosis virus. 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 present invention aims to provide a primer-probe combination and a kit for on-site detection of koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus. The primer-probe combination and the kit of the present invention are used to detect koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, with good sensitivity and accuracy, and the method is simple to operate, rapid, 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 koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus. The primer-probe combination comprises: primer pairs and probes for detecting koi herpesvirus, whose sequences are respectively shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting carp spring virus, whose sequences are respectively shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting carp edema virus, whose sequences are respectively shown in SEQ ID NOs: 7-9; primer pairs and probes for detecting goldfish hematopoietic necrosis virus, 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, BODIPYFL, 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 ATTO 425, 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 koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus.

[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 koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, 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 koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, the method comprising preparation of an integrated closed microfluidic chip kit, sample pretreatment, 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 a sample infected with koi herpes virus, carp spring virus, carp edema virus, or goldfish hematopoietic necrosis virus 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.

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

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

[0039] 2) Scan the QR code;

[0040] 3) Inserting 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 the use of the above-mentioned primer-probe combination or the above-mentioned kit or the above-mentioned method in detecting fish infected with koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus.

[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 koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus. The method for detecting koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus 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, rapid, and time-saving. The method provided by the present invention can simultaneously detect four viruses: koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, and plays an important role in on-site rapid detection of viral infections in aquaculture fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the sensitivity test result of Koi herpes virus.

[0053] Figure 2 This is the sensitivity test result of carp spring virus.

[0054] Figure 3 This is the sensitivity test result of carp edema virus.

[0055] Figure 4 This is the sensitivity test result of goldfish hematopoietic necrosis virus.

[0056] Figure 5 This is the actual sensitivity test result diagram.

[0057] Figure 6 This is a physical picture of the integrated microfluidic chip and sample processing tube.

[0058] Figure 7 This is the test result of actual fish tissue samples infected with Koi Herpes Virus.

[0059] Figure 8 This is the test result of actual fish tissue samples infected with carp spring virus.

[0060] Figure 9 This is the test result of actual fish tissue samples infected with carp edema virus.

[0061] Figure 10 This is the test result of actual fish tissue samples infected with goldfish hematopoietic necrosis virus.

[0062] Figure 11 This is a graph showing the detection results of Koi Herpes Virus in Comparative Example 1.

[0063] Figure 12 This is the test result diagram of carp spring virus in comparative example 1.

[0064] Figure 13 This is the test result diagram of carp edema virus in comparative example 1.

[0065] Figure 14 This is the test result of goldfish hematopoietic necrosis virus in comparative example 1. DETAILED DESCRIPTION

[0066] 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.

[0067] 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 6 shown.

[0068] The fish infected with koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus in the following examples were provided by the Animal Inspection Institute of the China Customs Science and Technology Research Center, a cooperative unit.

[0069] Example 1 Primer probe combination for rapid on-site detection of koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus

[0070] Primer and probe sequences were designed based on the specific sequences of koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus.

[0071] Among them, the primer probe sequence of Koi herpes virus is:

[0072] KHV-F:5'-GGACTGCTCGCTTAAAACC-3'(SEQ ID NO:1);

[0073] KHV-R: 5'-GACGAGTGCCGTCAAGCTA-3' (SEQ ID NO: 2);

[0074] KHV-P: 5'-ACATCAACGCACAACAATGCT-3' (SEQ ID NO: 3).

[0075] The primer and probe sequences for carp spring virus are:

[0076] SVCV-F:5'-TACCATAGATGAATGCAGG-3' (SEQ ID NO:4);

[0077] SVCV-R:5'-ACCCCAGATCTTCATCAGT-3' (SEQ ID NO:5);

[0078] SVCV-P: 5'-ATCATTCAAAGGATTGCATCAGG-3' (SEQ ID NO: 6).

[0079] The primer and probe sequences for carp edema virus are:

[0080] CEV-F:5'-GAGCAAGATTAGAAGTCA-3' (SEQ ID NO:7);

[0081] CEV-R:5'-ATACGTTGATACAATTCCA-3'(SEQ ID NO:8);

[0082] CEV-P: 5'-ATTGCAAATGTTATGTTCTGG-3' (SEQ ID NO: 9).

[0083] The primer and probe sequences for goldfish hematopoietic necrosis virus are:

[0084] GHFNV-F:5'-GGCCGCCAGTAACATGGAA-3' (SEQ ID NO:10);

[0085] GHFNV-R:5'-ACCTCTCGGTTCTTGTAGC-3' (SEQ ID NO:11);

[0086] GHFNV-P: 5'-CACAGCGAAGACCTGTACACGAT-3' (SEQ ID NO: 12).

[0087] The primer and probe sequences for the internal process control (IPC) are:

[0088] IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO: 13);

[0089] IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO: 14);

[0090] IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO: 15).

[0091] The primer-probe composition of koi herpesvirus, carp spring virus, carp edema virus and goldfish hematopoietic necrosis virus of the present invention is used for jointly or individually detecting koi herpesvirus, carp spring virus, carp edema virus and goldfish hematopoietic necrosis virus.

[0092] In the embodiment, the KHV probe is labeled with ATTO425 and the quenching group is SQ1, the SVCV probe is labeled with FAM and the quenching group is SQ1, the CEV probe is labeled with HEX and the quenching group is MGB, the GHFNV probe is labeled with CY5 and the quenching group is SQ1, and the IPC probe is labeled with CY5.5 and the quenching group is BHQ2.

[0093] Example 2 A microfluidic chip kit for detecting koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus

[0094] (1) The kit includes the primer-probe combination described in Example 1, with final concentrations as shown in Table 1 below.

[0095] Table 1 Primer and probe concentrations

[0096] Primer probe name Final concentration KHV-F, KHV-R 0.6μM, 0.6μM KHV-P 0.3 μM SVCV-F, SVCV-R 0.8μM, 0.8μM SVCV-P 0.4 μM CEV-F, CEV-R 0.8μM, 0.8μM CEV-P 0.4 μM GHFNV-F, GHFNV-R 0.4μM, 0.4μM GHFNV-P 0.2μM IPC-F, IPC-R 0.2μM, 0.2μM IPC-P 0.2μM

[0097] (2) The kit also includes purification reagents and air-dried reagents.

[0098] 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.

[0099] 2) The 50 μL air-dried reagent system is shown in Table 2 below.

[0100] Table 2 Air-drying reagent system

[0101] Element volume <![CDATA[4×Air-Dryable TM Direct RNA / DNA qPCR Mix]]> 12.5μL Primer probe mixture 2.5 μL

[0102] 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.

[0103] 3) The freeze-dried IPC quality control product includes the preparation of IPC pseudovirus and a freeze-dried reagent system.

[0104] According to the pseudovirus preparation method described in Example 1 of patent CN202110669475.X, a pseudovirus of the IPC gene was constructed and obtained.

[0105] The concentration was 1×10 6 copies / mL IPC pseudovirus 5 μL was prepared into freeze-dried pellets using a common freeze-drying system and method;

[0106] Table 3 Lyophilized IPC reagent system

[0107] Element content IPC pseudovirus <![CDATA[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％

[0108] 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.

[0109] Table 4 Freeze-drying procedure

[0110]

[0111] The freeze-dried balls are placed on the chip with air-dried reaction reagents to assemble an integrated detection kit.

[0112] Example 3 Positive sample preparation

[0113] 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.

[0114] Table 5 Target information

[0115] Target information copies / ng Length (bp) Carp Spring Virus (Carp) 2.75E+08 3314 Koi herpes virus (carp) 2.79E+08 3276 Goldfish hematopoietic necrosis virus (carp) 2.71E+08 3364 Cyprinus cyprinid edema virus (carp) 2.80E+08 3264

[0116] Example 4 Preparation of sample processing tube

[0117] (1) Install the sample processing tube;

[0118] (2) Add about 1 g of corundum 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;

[0119] (3) Place the tube in the tooling position of a precision manual press and wrap it in 160°C aluminum foil.

[0120] Example 5 Detection Method for Koi Herpes Virus, Carp Spring Virus, Carp Edema Virus, and Goldfish Hematopoietic Necrosis Virus

[0121] (1) Remove the aluminum foil protective film from the sample processing tube, place it vertically, add the shrimp to be tested into the sample processing tube, and tighten the tube cap;

[0122] (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;

[0123] (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;

[0124] (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;

[0125] (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;

[0126] (6) After waiting for 3 seconds, you will see the red light flashing in the scanning window and scan the chip QR code;

[0127] (7) Scan the sample information or skip according to the on-screen prompts;

[0128] (8) Follow the on-screen instructions to open the hatch, insert the chip, and close the hatch;

[0129] (9) Click the "Run" button to start the test;

[0130] (10) After the operation is completed, check the test results.

[0131] Sample processing time in the sample processing 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 program 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 detecting koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, from sample processing, nucleic acid extraction and purification, to amplification and test results, takes approximately 37 minutes.

[0132] Experimental Example 1 Sensitivity Detection

[0133] Samples containing 1000, 500, and 100 copies of koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus plasmid were prepared for nucleic acid amplification detection. The system components are shown in Table 2, and the test results are shown in Table 6 below. The detection curve is shown in Table 6 below. Figures 1-4 shown.

[0134] Table 6 Sensitivity test results

[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 containing 100 copies of koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus.

[0137] Experimental Example 2: Actual Sample Testing

[0138] Fish meat (0.5 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 7-10 shown.

[0140] Table 7 Sample test results

[0141] Virus type Target Ct value IPC internal reference Ct value Test results Koi herpes virus 33.06 30.85 Positive Carp Spring Virus 36.40 31.12 Positive Carp edema virus 33.59 31.12 Positive Goldfish hematopoietic necrosis virus 33.52 31.48 Positive

[0142] It can be seen from Table 7 that the primer-probe composition for koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus 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 probe sequence of Koi herpes virus is:

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

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

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

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

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

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

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

[0153] The primer and probe sequences for carp edema virus are:

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

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

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

[0157] The primer and probe sequences for goldfish hematopoietic necrosis virus are:

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

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

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

[0161] The experimental results of Experimental Example 2 and Comparative Example 1 are as follows: Figure 11-14 As shown in Table 8, the Ct values ​​are as follows:

[0162] Table 8 Sample test results

[0163] Virus type Experimental Example 2 Ct value Comparative Example 1 Ct value Koi herpes virus 33.06 34.42 Carp Spring Virus 36.40 36.29 Carp edema virus 33.59 34.39 Goldfish hematopoietic necrosis virus 33.52 (-)

[0164] As can be seen from the figure, the fluorescence values ​​are all at a low level, and some targets are not amplified, indicating that the 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 koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, characterized in that: The primer-probe combination includes: primer pairs and probes for detecting koi herpes virus, whose sequences are respectively shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting carp spring virus, whose sequences are respectively shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting carp edema virus, whose sequences are respectively shown in SEQ ID NOs: 7-9; primer pairs and probes for detecting goldfish hematopoietic necrosis virus, 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.

2. Use of the primer-probe combination according to claim 1 in preparing products for detecting koi herpesvirus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus.

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 koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, 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 koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus, 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 koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus 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 tissue infected with koi herpes virus, carp spring virus, carp edema virus, and goldfish hematopoietic necrosis virus.

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) Insert 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 koi herpesvirus, carp spring virus, carp edema virus, or goldfish hematopoietic necrosis virus.

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