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

Abstract

The invention discloses a primer probe combination and a kit for detecting shrimp viruses on site, and relates to the technical field of gene detection. The invention provides a primer probe combination for detecting enterocytozoon hepatopenaei, prawn white spot syndrome virus and macrobrachium rosenbergii nodavirus on site. The primer probe combination comprises a primer pair and a probe for detecting enterocytozoon hepatopenaei, the primer pair and the probe are used for detecting the prawn white spot syndrome virus; the invention discloses a primer pair and a probe for detecting macrobrachium rosenbergii nodavirus. The method for detecting the shrimp 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 shrimp viruses.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, in particular to a primer probe combination and a kit for on-site detection of shrimp hepatocellular carcinoma, white spot syndrome virus of shrimp and Macrobrachium rosenbergii Nodavirus. Background Art

[0002] Enterocytozoon hepatopenaei (EHP) is a highly contagious parasitic disease of shrimp. Infection is caused by the parasite Enterocytozoon hepatopenaei (EHP) and is a common disease of shrimp. In the early stages, infected shrimp show no obvious symptoms. In the middle stages, individuals move slowly and stay at the bottom, often surfacing in the early morning. They also eat less, react slowly, and have a decreased ability to cope with stress. In the later stages, diseased shrimp may vary in size within the group.

[0003] White Spot Syndrome Virus (WSSV) is a shrimp disease caused primarily by the White Spot Syndrome Virus (WSSV). Infected shrimp develop white spots on the inside of their carapaces, and the cephalothorax separates from the underlying tissue, making it easy to peel off. Sick shrimp typically stop eating, become sluggish, have weak limbs, have dull eyes, and have pale abdominal muscles and a reddish skin. They swim at the surface or lie motionless at the edge of the pond, eventually dying.

[0004] Macrobrachium rosenbergii, also known as the Malaysian prawn, is a major aquaculture species of freshwater crustacean in my country. my country's aquaculture area for Macrobrachium rosenbergii exceeds 500,000 mu (approximately 16,000 hectares), with a total production exceeding 100,000 tons, making it a global leader in the cultivation of Macrobrachium rosenbergii. Muscle leucorrhea (Muscle turbidity) is a new disease that has recently emerged in major production areas of Macrobrachium rosenbergii in my country. Also known as Macrobrachium rosenbergii white body disease or white tail disease, the pathogen has been identified as the Macrobrachium rosenbergii Nodavirus. This disease primarily affects desalinated Macrobrachium rosenbergii seedlings. Diseased shrimp exhibit white spots, turbidity, or a general whitening of the muscles. Sick shrimp can die within a few days, with mortality rates as high as 40% to 90%.

[0005] Infection of aquaculture with these viruses will greatly increase the cost of aquaculture and cause incalculable losses to the aquaculture industry.

[0006] Currently available methods for detecting these viruses include enzyme-linked immunosorbent assays (ELISAs) and nucleic acid detection methods. For example, patent CN118745215A discloses a method for detecting shrimp hepatocellular carcinoma using colloidal gold, while patent CN101875980A discloses a method for detecting Macrobrachium rosenbergii Nodavirus. These methods can detect shrimp hepatocellular carcinoma or Macrobrachium rosenbergii Nodavirus alone, but lack detection capabilities for the other two viruses.

[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. The present invention, based on the sample processing and detection equipment, 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 three pathogens, namely shrimp hepatocellular carcinoma, shrimp white spot syndrome virus, and Macrobrachium rosenbergii nodavirus. 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 shrimp hepatocellular carcinoma, shrimp white spot syndrome virus, and Macrobrachium rosenbergii nodavirus. The primer-probe combination and the kit of the present invention are used to detect shrimp hepatocellular carcinoma, shrimp white spot syndrome virus, and Macrobrachium rosenbergii nodavirus, 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] In one aspect, the present invention provides a primer-probe combination for on-site detection of Enterocytosis, white spot syndrome virus, and Macrobrachium rosenbergii nodavirus. The primer-probe combination comprises: a primer pair and a probe for detecting Enterocytosis, whose sequences are represented by SEQ ID NOs: 1-3; a primer pair and a probe for detecting White spot syndrome virus, whose sequences are represented by SEQ ID NOs: 4-6; a primer pair and a probe for detecting Macrobrachium rosenbergii nodavirus, whose sequences are represented by SEQ ID NOs: 7-9; and a primer pair and a probe for detecting IPC, whose sequences are represented by SEQ ID NOs: 10-12.

[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 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 a product for detecting shrimp hepatocellular carcinoma, white spot syndrome virus of shrimp, and Macrobrachium rosenbergii Nodavirus.

[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 shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus, wherein the kit comprises 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 shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii nodavirus, 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 shrimp hepatocellular carcinoma, white spot syndrome virus, or Macrobrachium rosenbergii Nodavirus to 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 described in step (2) is shrimp such as prawns and Macrobrachium rosenbergii.

[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 primer-probe combination, the above kit, or the above method in detecting shrimps that are infected with Enterocytozoa hepatocellularis, white spot syndrome virus, or Macrobrachium rosenbergii Nodavirus.

[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 shrimp hepatocellular carcinoma, white spot syndrome virus (WSSV), and Macrobrachium rosenbergii nodavirus. The method for detecting shrimp hepatocellular carcinoma, shrimp white spot syndrome virus, and Macrobrachium rosenbergii nodavirus using the primer-probe combination and kit of the present invention integrates sample pretreatment, nucleic acid extraction, purification, amplification, and detection, exhibiting good sensitivity and accuracy. The method is simple to operate, rapid, and time-saving. The method provided by the present invention can simultaneously detect shrimp hepatocellular carcinoma, shrimp white spot syndrome virus, and Macrobrachium rosenbergii nodavirus, playing an important role in the on-site rapid detection of viral infections in aquaculture shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the sensitivity test result of shrimp hepatocellular carcinoma.

[0053] Figure 2 This is the sensitivity test result of white spot syndrome virus in shrimp.

[0054] Figure 3 This is the sensitivity test result of Macrobrachium rosenbergii Nodavirus.

[0055] Figure 4 This is a physical picture of the integrated microfluidic chip and sample processing tube.

[0056] Figure 5 This is the test result of actual shrimp meat samples infected with Enterocytosis.

[0057] Figure 6 This is the test result of actual shrimp viscera samples infected with shrimp hepatocellular carcinoma.

[0058] Figure 7 This is the test result of actual shrimp head tissue sample infected with shrimp hepatoenterozoa.

[0059] Figure 8 This is the test result of actual shrimp meat samples infected with white spot syndrome virus.

[0060] Figure 9 This is the test result of actual samples of shrimp viscera infected with white spot syndrome virus.

[0061] Figure 10 This is the test result of actual sample of shrimp head tissue infected with white spot syndrome virus.

[0062] Figure 11 This is a picture showing the test results of actual shrimp meat samples infected with Macrobrachium rosenbergii Nodavirus.

[0063] Figure 12 This is a picture of the test results of actual shrimp viscera samples infected with Macrobrachium rosenbergii Nodavirus.

[0064] Figure 13 This is the test result of actual sample of shrimp head tissue infected with Macrobrachium rosenbergii Nodavirus.

[0065] Figure 14 This is the result of testing the white spot syndrome virus in shrimp in comparative example 1.

[0066] Figure 15 This is the result of the Nodavirus test for Macrobrachium rosenbergii in comparative example 1. DETAILED DESCRIPTION

[0067] 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.

[0068] 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 4 shown.

[0069] In the following examples, the shrimp and Macrobrachium rosenbergii infected with Enterocytosis hepatocellularis, White Spot Syndrome Virus, and Macrobrachium rosenbergii Nodavirus were provided by the Animal Inspection Institute of the China Customs Science and Technology Research Center, a cooperative unit.

[0070] Example 1 Primer probe combination for rapid on-site detection of shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus

[0071] Primer and probe sequences were designed based on the specific sequences of shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus.

[0072] Among them, the primer probe sequence of shrimp hepatocystis is:

[0073] EHP-F:5'-TTCTCAAACATTTTCACCATTGGT-3' (SEQ ID NO:1);

[0074] EHP-R:5'-CTGTGTCTGTGTAAATATCGTCTCTTTTT-3'(SEQ ID NO:2);

[0075] EHP-P: 5'-ATTTCAAACACTGTAAACCT-3' (SEQ ID NO: 3).

[0076] The primer and probe sequences for white spot syndrome virus are:

[0077] WSSV-F:5'-CAACCTCGACGTTATTGTCAAACC-3' (SEQ ID NO:4);

[0078] WSSV-R:5'-GCTGAAGCGACAGTTCCCT-3' (SEQ ID NO:5);

[0079] WSSV-P: 5'-ACGTGCTGGCATGACATTCCAA-3' (SEQ ID NO: 6).

[0080] The primer and probe sequences for Macrobrachium rosenbergii Nodavirus are:

[0081] MrNV-F:5'-GTAGCAACACTAAAGGGAG-3'(SEQ ID NO:7);

[0082] MrNV-R:5'-TTGCTGCTGATAGAACGTCC-3'(SEQ ID NO:8);

[0083] MrNV-P: 5'-GTTCCGGGATTTACGGCTT-3' (SEQ ID NO: 9).

[0084] The primer and probe sequences for the internal process control (IPC) are:

[0085] IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO: 10);

[0086] IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO: 11);

[0087] IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO: 12).

[0088] The primer-probe combination for shrimp hepatocystis, white spot syndrome virus and Macrobrachium rosenbergii nodavirus of the present invention is used for the joint or separate detection of shrimp hepatocystis, white spot syndrome virus and Macrobrachium rosenbergii nodavirus.

[0089] In the embodiment, the EHP probe is labeled with FAM and the quencher group is MGB, the WSSV probe is labeled with HEX and the quencher group is SQ1, the MrNV probe is labeled with CY5 and the quencher group is BHQ2, and the IPC probe is labeled with CY5.5 and the quencher group is BHQ2.

[0090] Example 2 A microfluidic chip kit for detecting shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus

[0091] (1) The kit includes the primer-probe combination described in Example 1, with final concentrations as shown in Table 1 below.

[0092] Table 1 Primer and probe concentrations

[0093] Primer probe name Final concentration EHP-F, EHP-R 0.8μM, 0.8μM EHP-P 0.15μM WSSV-F, WSSV-R 0.6μM, 0.6μM WSSV-P 0.2μM MrNV-F, MrNV-R 0.6μM, 0.6μM MrNV-P 0.3 μM IPC-F, IPC-R 0.2μM, 0.2μM IPC-P 0.2μM

[0094] (2) The kit also includes purification reagents and air-dried reagents.

[0095] 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.

[0096] 2) The 50 μL air-dried reagent system is shown in Table 2 below.

[0097] Table 2 Air-drying reagent system

[0098] Element volume <![CDATA[4×Air-Dryable TM Direct RNA / DNA qPCR Mix]]> 12.5μL Primer probe mixture 2.5 μL

[0099] 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.

[0100] 3) The freeze-dried IPC quality control product includes the preparation of IPC pseudovirus and a freeze-dried reagent system.

[0101] According to the pseudovirus preparation method described in Example 1 of patent CN202110669475.X, a pseudovirus of the IPC gene was constructed and obtained.

[0102] 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;

[0103] Table 3 Lyophilized IPC reagent system

[0104]

[0105]

[0106] 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.

[0107] Table 4 Freeze-drying procedure

[0108]

[0109] The freeze-dried balls are placed on the chip with air-dried reaction reagents to assemble an integrated detection kit.

[0110] Example 3 Positive sample preparation

[0111] 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 the table below.

[0112] Table 5 Target information

[0113] Target information copies / ng Length (bp) White spot syndrome virus (shrimp) 2.73E+08 3337 Macrobrachium rosenbergii Nodavirus (shrimp) 2.82E+08 3235 Shrimp Hepatocellular Carcinoma (Shrimp) 2.84E+08 3210

[0114] Example 4 Preparation of sample processing tube

[0115] (1) Install the sample processing tube;

[0116] (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;

[0117] (3) Place the tube in the tooling position of a precision manual press and wrap it in 160°C aluminum foil.

[0118] Example 5 Detection Methods for Enterocytosis, White Spot Syndrome Virus, and Macrobrachium rosenbergii Nodavirus

[0119] (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;

[0120] (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;

[0121] (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;

[0122] (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;

[0123] (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;

[0124] (6) After waiting for 3 seconds, you will see the red light flashing in the scanning window and scan the chip QR code;

[0125] (7) Scan the sample information or skip according to the on-screen prompts;

[0126] (8) Follow the on-screen instructions to open the hatch, insert the chip, and close the hatch;

[0127] (9) Click the "Run" button to start the test;

[0128] (10) After the operation is completed, check the test results.

[0129] 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 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 of 19 minutes. The entire process for detecting Enterocytosis hepatocellularis, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus, from sample processing, nucleic acid extraction and purification, to amplification and detection results, takes approximately 37 minutes.

[0130] Experimental Example 1 Sensitivity Detection

[0131] The content of shrimp hepatocellular carcinoma, white spot syndrome virus and Macrobrachium rosenbergii Noda virus plasmid is 5×10 5 , 5×10 4 , 5×10 3 The samples with the highest copy number were subjected to nucleic acid amplification test. The system source components are shown in Table 2. The test results are shown in Table 6 below. The test curve is shown in Figure 1-Figure 3 shown.

[0132] Table 6 Sensitivity test results

[0133]

[0134] As shown in Table 6 above, the primer combination and kit described in this application have good sensitivity and can detect the concentration of 5×10 2The copy numbers of plasmid samples of Enterocytozoa, White Spot Syndrome Virus and Macrobrachium rosenbergii Nodavirus were detected.

[0135] Experimental Example 2: Actual Sample Testing

[0136] Shrimp meat (0.5 g), shrimp viscera (0.2 g), and shrimp head tissue (0.5 g) infected with different types of viruses were used in the experiment.

[0137] 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 5-Figure 13 shown.

[0138] Table 7 Sample test results

[0139]

[0140]

[0141] Table 7 shows that the primer-probe combination for Enterocytosis hepatocellularis, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus of the present invention can detect different parts of real samples and can be used for on-site rapid identification.

[0142] Comparative Example 1

[0143] 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:

[0144] Among them, the primer probe sequence of white spot syndrome virus is:

[0145] WSSV-F:5'-CCGACGCCAAGGGAACT-3'(SEQ ID NO:13);

[0146] WSSV-R:5'-TTCAGATTCGTTACCGTTTCCA-3' (SEQ ID NO:14);

[0147] WSSV-P: 5'-CGCTTCAGCCATGCCAGCCG-3' (SEQ ID NO: 15).

[0148] The primer and probe sequences for Macrobrachium rosenbergii Nodavirus are:

[0149] MrNV-F:5'-GACCCAAAAGTAGCGAAGGA-3' (SEQ ID NO:16);

[0150] MrNV-R:5'-GGCYTCTCCCTTTAGTGTT-3' (SEQ ID NO:17);

[0151] MrNV-P: 5'-AAGCAACCGCCTTCAATGCC-3' (SEQ ID NO: 18).

[0152] White spot syndrome virus, Macrobrachium rosenbergii Noda virus plasmid content is 5×10 5 The experimental results of the experimental example and comparative example 1 are as follows Figure 14-15 As shown in Table 7, the detection Ct values ​​are as follows:

[0153] Table 7 Comparative Example 1 Detection Results Ct Value

[0154] Virus type Experimental Example 1 Ct value Comparative Example 1 Ct value White spot syndrome virus 28.34 38.62 Macrobrachium rosenbergii Nodavirus 26.97 36.34

[0155] As can be seen from the figure, the Ct values ​​are delayed to a certain extent and the fluorescence values ​​are also low, indicating that the detection sensitivity is low.

[0156] 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 shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus, characterized in that: The primer-probe combination includes: primer pairs and probes for detecting shrimp hepatocellular carcinoma, whose sequences are respectively shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting white spot syndrome virus of shrimp, whose sequences are respectively shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting Macrobrachium rosenbergii Nodavirus, whose sequences are respectively shown in SEQ ID NOs: 7-9; and primer pairs and probes for detecting IPC, whose sequences are respectively shown in SEQ ID NOs: 10-12.

2. Use of the primer-probe combination according to claim 1 in the preparation of a product for detecting shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus.

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 shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus, 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 shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus, 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 shrimp hepatocellular carcinoma, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus to 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 shrimps such as shrimps infected with enterocytosis, white spot syndrome virus, and Macrobrachium rosenbergii Nodavirus.

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 shrimp infected with Enterocytosis hepatocellularis, white spot syndrome virus, or Macrobrachium rosenbergii Nodavirus.

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