Primer probe combination and kit for detecting prawn hepatopancreatic parvovirus and application of primer probe combination and kit

Abstract

The invention discloses a primer probe combination and a kit for detecting prawn hepatopancreatic parvovirus and application of the primer probe combination and the kit. The primer probe combination comprises an outer primer pair F3 and B3, an inner primer pair FIP and BIP and a molecular beacon probe MB. The primer probe combination for detecting the prawn hepatopancreatic parvovirus, provided by the invention, has the characteristics of high sensitivity, strong specificity, good stability and good repeatability, and can be used for specifically detecting the prawn hepatopancreatic parvovirus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a primer-probe combination, a kit and their applications for detecting Hepatopancreatic parvovirus (HPV) of shrimps. Background Art

[0002] Hepatopancreatic parvovirus (HPV) is a single-stranded DNA virus, spherical in shape, with a diameter of 22-24 nm, and is one of the important pathogens causing diseases in shrimps. The virus attacks during the shrimp seedling stage. After the larvae are infected, they will show symptoms such as slow growth, soft carapace, and poor stress resistance, which easily lead to secondary bacterial infections in young shrimps. The infection rate of shrimps can reach 70%-80%, and the disease condition is more severe during the larval stage, with a mortality rate of 50%-90%. It is a very harmful disease. Since the infected shrimps do not have specific external symptoms and often suffer from other bacterial diseases at the same time, farmers cannot quickly determine whether there is a virus infection, resulting in untimely implementation of relevant prevention and control measures and missed treatment time, causing economic losses. In view of the above situation, it is very necessary to study an early and rapid diagnosis method for this disease.

[0003] Currently, the methods for detecting this disease mainly include pathological section detection, PCR, qPCR, etc. Among them, pathological section detection uses the pathological signs of the diseased tissues for detection, with cumbersome operation steps and long time consumption. Compared with pathological sections, the operation steps of PCR detection are simplified, but gel electrophoresis is required, which is likely to cause environmental pollution. The qPCR detection method is carried out under closed conditions throughout the process, which can avoid contamination and has the advantages of high sensitivity and strong specificity. However, this detection method can only be carried out in the laboratory and requires professional technical personnel and expensive temperature control equipment to achieve detection, and the temperature-variable amplification takes a long time, which is not conducive to rapid on-site detection. Therefore, an accurate and rapid detection method is needed. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a primer-probe combination for detecting Hepatopancreatic parvovirus of shrimps.

[0005] The present invention also provides a kit containing the above primer-probe combination.

[0006] The present invention also provides the applications of the above primer-probe combination and kit.

[0007] The present invention also provides a method for detecting Hepatopancreatic parvovirus of shrimps for non-diagnostic purposes.

[0008] According to an embodiment of the first aspect of the present invention, a primer-probe combination for detecting Taura syndrome virus is proposed. The primer-probe combination includes an outer primer pair F3 and B3, an inner primer pair FIP and BIP, and a molecular beacon probe MB;

[0009] The nucleic acid sequences of the outer primer pair F3 and B3 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively;

[0010] The nucleic acid sequences of the inner primer pair FIP and BIP are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively;

[0011] The nucleic acid sequence of the molecular beacon probe MB is shown in SEQ ID NO: 5.

[0012] The primer-probe combination for detecting Taura syndrome virus according to the embodiment of the present invention has at least the following beneficial effects:

[0013] The primer-probe combination for detecting Taura syndrome virus provided by the present invention has the characteristics of high sensitivity, strong specificity, good stability and good repeatability, and can be used for specific detection of Taura syndrome virus.

[0014] According to some embodiments of the present invention, the 5' end of the molecular beacon probe MB is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group. In the LAMP experiment, when the molecular beacon probe MB approaches the target DNA sequence to form a hybrid DNA-RNA strand, its hairpin structure is destroyed; the ribonuclease of RNase HII can specifically recognize the DNA-RNA pairing and hydrolyze the phosphodiester bond of RNA to generate a free 3'-OH end; the cleaved probe can serve as a new primer to further accelerate the amplification process, which is faster than the ordinary MB-LAMP detection and has better repeatability.

[0015] According to some embodiments of the present invention, the fluorescent reporter group includes one of FAM, TET, JOE, Cy3, Cy5, Cy5.5, Lluorescein, Rhodamine, Rhodamine Red, Rhodamine 6G, Orengon Green 488, OrengonGreen500, Orengon Green 514, Texas Red, TAMRA, Inosine, HEX, FITC, Acridine orange and ROX.

[0016] According to some embodiments of the present invention, the fluorescent quenching group includes one of Dabcyl, DABSYL, TAMRA, BHQ-1, BHQ-2 and BHQ-3.

[0017] According to some embodiments of the present invention, the 9 bases at the 5'-end and the 9 bases at the 3'-end of the molecular beacon probe MB are completely complementary to form a stem-loop structure.

[0018] According to some embodiments of the present invention, there are five consecutive ribonucleotides at positions 20bp - 24bp of the sequence shown in SEQ ID NO: 5.

[0019] According to some embodiments of the present invention, the molecular beacon probe MB is designed at the LAMP loop primer.

[0020] According to some embodiments of the present invention, the nucleotide sequence of the LAMP loop primer is as shown in SEQ ID NO: 6.

[0021] According to the embodiments of the second aspect of the present invention, a kit is provided, which includes the primer-probe combination described in the embodiments of the first aspect of the present invention. Since the kit of the embodiments of the present invention adopts all the technical solutions of the primer-probe combination of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments. At the same time, the kit provided by the present invention can not only detect the amplification result in real time through a thermostatic fluorescence detector, but also judge the amplification result by irradiating the amplification product under an ultraviolet lamp when the conditions are simple. It has the characteristics of high sensitivity, high specificity and high stability, and is suitable for laboratory and on-site detection.

[0022] According to some embodiments of the present invention, the kit further includes at least one of a positive control, a negative control, and a LAMP reaction premix.

[0023] According to some embodiments of the present invention, the LAMP reaction premix includes LAMP Buffer, Bst enzyme, and RNaseHⅡ.

[0024] According to some embodiments of the present invention, the LAMP Buffer includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, 20, dNTP.

[0025] According to some embodiments of the present invention, the positive control includes a recombinant vector containing a nucleic acid molecule of Taura syndrome virus.

[0026] According to some embodiments of the present invention, the negative control includes water.

[0027] According to the embodiments of the third aspect of the present invention, the above primer-probe combination or kit is applied in any one of the following:

[0028] 1) Detection for non-diagnostic purposes or auxiliary detection of Taura syndrome virus;

[0029] 2) Prepare a product for detecting or assisting in the detection of Taura syndrome virus.

[0030] According to some embodiments of the present invention, the product is selected from reagents, kits or chips.

[0031] According to some embodiments of the present invention, the method of using the product includes the following steps:

[0032] Use the above primer-probe combination or kit to detect the DNA of the sample to be tested.

[0033] According to some embodiments of the present invention, the detection includes using the DNA of the sample to be tested as a template, performing fluorescence loop-mediated isothermal amplification using the above primer-probe combination or kit, monitoring the fluorescence signal, and determining the result.

[0034] According to some embodiments of the present invention, the reaction system for fluorescence loop-mediated isothermal amplification includes:

[0035]

[0036]

[0037] According to some embodiments of the present invention, the reaction system for fluorescence loop-mediated isothermal amplification further includes water.

[0038] According to some embodiments of the present invention, the reaction system for fluorescence loop-mediated isothermal amplification further includes template DNA, and the addition amount of the template DNA is 4-6 μL.

[0039] According to some embodiments of the present invention, the fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument or a constant temperature device.

[0040] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument, the result determination is as follows: If a positive amplification curve is shown, the sample to be tested is positive and contains Taura syndrome virus; if a positive amplification curve is not shown, the sample to be tested is negative and does not contain Taura syndrome virus.

[0041] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument, the amplification program is: 58-65 °C for 0.5-2 min, 40-48 cycles, and fluorescence data is collected.

[0042] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument, the amplification program is: 62 °C for 1 min, 45 cycles, and fluorescence data is collected.

[0043] According to some embodiments of the present invention, the thermostatic device includes a thermostat.

[0044] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is carried out using a thermostatic device, the amplification program is: 58 - 65°C for 40 - 50 min.

[0045] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is carried out using a thermostatic device, the amplification program is: 62°C for 45 min.

[0046] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is carried out using a thermostatic device, the result determination is as follows: Place the product obtained by the fluorescence loop-mediated isothermal amplification under ultraviolet light irradiation. If there is an obvious fluorescence signal, the test sample is positive and contains Taura syndrome virus; if there is no fluorescence signal, the test sample is negative and does not contain Taura syndrome virus.

[0047] According to the embodiments of the fourth aspect of the present invention, a method for detecting or assisting in the detection of Taura syndrome virus for non-diagnostic purposes is provided, including the following steps:

[0048] Detect the DNA of the test sample using the above primer-probe combination or kit.

[0049] According to some embodiments of the present invention, the detection includes using the DNA of the test sample as a template, performing fluorescence loop-mediated isothermal amplification using the above primer-probe combination or kit, monitoring the fluorescence signal, and determining the result.

[0050] According to some embodiments of the present invention, the reaction system of the fluorescence loop-mediated isothermal amplification includes:

[0051]

[0052] According to some embodiments of the present invention, the reaction system of the fluorescence loop-mediated isothermal amplification further includes water.

[0053] According to some embodiments of the present invention, the reaction system of the fluorescence loop-mediated isothermal amplification further includes template DNA, and the addition amount of the template DNA is 4 - 6 μL.

[0054] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is carried out using a qPCR instrument or a thermostatic device.

[0055] According to some embodiments of the present invention, when the fluorescence loop-mediated isothermal amplification is carried out using a qPCR instrument, the determination is as follows: If a positive amplification curve is shown, the test sample is positive and contains Taura syndrome virus; if a positive amplification curve is not shown, the test sample is negative and does not contain Taura syndrome virus.

[0056] According to some embodiments of the present invention, when the fluorescent loop-mediated isothermal amplification is performed using a qPCR instrument, the amplification program is as follows: 58 - 65°C for 0.5 - 2 min, 40 - 48 cycles, and fluorescence data is collected.

[0057] According to some embodiments of the present invention, when the fluorescent loop-mediated isothermal amplification is performed using a qPCR instrument, the amplification program is as follows: 62°C for 1 min, 45 cycles, and fluorescence data is collected.

[0058] According to some embodiments of the present invention, the constant temperature device includes a thermostatic instrument.

[0059] According to some embodiments of the present invention, when the fluorescent loop-mediated isothermal amplification is performed using a constant temperature device, the amplification program is as follows: 58 - 65°C for 40 - 50 min.

[0060] According to some embodiments of the present invention, when the fluorescent loop-mediated isothermal amplification is performed using a constant temperature device, the amplification program is as follows: 62°C for 45 min.

[0061] According to some embodiments of the present invention, when the fluorescent loop-mediated isothermal amplification is performed using a constant temperature device, the result determination is as follows: The product obtained by the fluorescent loop-mediated isothermal amplification is placed under an ultraviolet lamp for irradiation. If there is an obvious fluorescence signal, the test sample is positive and contains Taura syndrome virus; if there is no fluorescence signal, the test sample is negative and does not contain Taura syndrome virus.

[0062] According to the embodiments of the fifth aspect of the present invention, a system for detecting Taura syndrome virus includes:

[0063] A detection module: used to perform fluorescent loop-mediated isothermal amplification on the nucleic acid of the test sample using the above primer-probe combination or kit;

[0064] An analysis module: used to evaluate whether the test sample contains Taura syndrome virus.

[0065] According to some embodiments of the present invention, the system further includes an extraction module for extracting the nucleic acid of the test sample.

[0066] Other features and advantages of the present invention will be described in the subsequent specification, and, in part, will be obvious from the specification, or will be understood by implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is a flow chart for the design and screening of the LAMP primer combination provided by the embodiments of the present invention;

[0068] Figure 2Specific structural diagram of the molecular beacon probe provided by the embodiment of the present invention;

[0069] Figure 3 Sensitivity detection result diagram provided by the embodiment of the present invention;

[0070] Figure 4 Specificity detection result diagram provided by the embodiment of the present invention;

[0071] Figure 5 Result diagram of detection using a thermostatic instrument provided by the embodiment of the present invention, where 1-8 are sample numbers. Detailed implementation manners

[0072] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where: The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0073] For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0074] Unless otherwise specified, nucleotide sequences are in the order of 5' to 3'.

[0075] Embodiment 1

[0076] This embodiment provides a reagent for detecting Taura syndrome virus, which consists of a LAMP primer combination and a molecular beacon probe. The design and screening flow chart of the LAMP primer combination is as Figure 1 shown, and the specific steps are as follows:

[0077] (1) Design of the LAMP primer combination

[0078] Through a large amount of literature retrieval and analysis, and at the same time through sequence retrieval and alignment analysis in the GenBank database, conserved sequences of the Taura syndrome virus genome were selected, and a series of LAMP primers were designed. After synthesis, they were screened and verified through experiments, and the sequences shown in Table 1 were preferably obtained, including the upstream outer primer F3, the downstream outer primer B3, the upstream inner primer FIP, and the downstream inner primer BIP.

[0079] Table 1

[0080]

[0081] (2) Design molecular beacon probes at the LAMP loop primers

[0082] The molecular beacon probes are designed at the LAMP loop primers (the LAMP loop primer LB sequence is: AGTGGCTTTCGTGGTGGC (SEQ ID NO: 6)). They have five consecutive ribonucleotides that can be cleaved by ribonuclease, simultaneously initiating rapid nucleic acid amplification and generating sequence-specific fluorescence. The specific sequence of the molecular beacon probes is shown in SEQ ID NO: 5. Among them, the underlined part “—” in the molecular beacon is the RNA sequence, and the 5'-end and 3'-end are respectively labeled with a fluorophore (JOE) and a quencher (Dabcyl). The 9 bases at the 5'-end and the 9 bases at the 3'-end are completely complementary, forming a stem-loop structure.

[0083] The specific structure of the molecular beacon probes is as shown in Figure 2 . In the LAMP experiment, when the probe approaches the target DNA sequence, a hybrid DNA-RNA strand is formed, and its hairpin structure is destroyed. The ribonuclease of RNase HII can specifically recognize the DNA-RNA pairing and hydrolyze the phosphodiester bond of RNA, generating a free 3'-OH end; the cleaved probe can serve as a new primer to further accelerate the amplification process, which is faster and has better repeatability compared to ordinary MB-LAMP detection.

[0084] Example 2

[0085] This example provides a kit for detecting Taura syndrome virus, which consists of 5×LAMP Buffer (purchased from Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.), Bst enzyme (purchased from Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.), RNase HⅡ (purchased from Guangzhou MegaGen Biotech Co., Ltd.), amplification primers (including upstream external primer F3, downstream external primer B3, upstream internal primer FIP, downstream internal primer BIP, the sequences are shown in Table 1), downstream loop primer molecular beacon probes (the sequences are shown in Table 1), positive control, and negative control;

[0086] Among them, the positive control product is a cloned plasmid containing the amplification target fragment of Taura syndrome virus synthesized by General Biosystems.

[0087] The negative control product is sterile ultrapure water.

[0088] Example 3

[0089] This example provides a method for detecting Taura syndrome virus, and the steps are as follows:

[0090] Extract the nucleic acid sample of the sample to be tested as a template, and use the kit prepared in Example 2 to prepare the MB-LAMP reaction system shown in Table 2 below. Perform LAMP amplification on the prepared MB-LAMP reaction system.

[0091] In the case of having qPCR instrument equipment, it can be selected to perform LAMP amplification on a qPCR instrument (such as SLAN full-automatic medical PCR analysis system, etc.) according to the procedure shown in Table 3 below.

[0092] In the case of only having a thermostatic incubator, it can be selected to perform LAMP amplification on a thermostatic incubator (such as Xiangyi dry-type thermostatic incubator) according to the procedure shown in Table 4 below.

[0093] Table 2

[0094]

[0095]

[0096] Table 3

[0097]

[0098] Table 4

[0099]

[0100] Result determination:

[0101] If detected by a qPCR instrument, a sample with an obvious S-shaped amplification curve in the HEX channel of the sample is judged as positive, and a sample without an amplification curve is judged as negative.

[0102] If detected by a thermostatic incubator, irradiate the amplification product under an ultraviolet lamp. A sample with obvious fluorescence signal is positive, and a sample without fluorescence signal is negative.

[0103] Example 4 Sensitivity experiment

[0104] To meet the requirements of the application scenario, the present invention extracts the nucleic acid from the hepatopancreas tissue of a shrimp confirmed to be infected with hepatopancreatic parvovirus of shrimp. The extraction kit uses Tiangen marine animal tissue genomic DNA kit (product number DP324), detects it by qPCR method, and calculates the content of its viral genomic DNA. Select a positive sample (CT value is 21.98), calculate the virus concentration as 39810 copies / μL, perform 10-fold serial dilution, use the series dilution samples as templates, use the kit prepared in Example 2, and the detection method provided in Example 3 for detection (select a qPCR instrument for LAMP amplification), until the signal of a certain dilution sample cannot be detected, and use this concentration as the detection limit of the sample to test the sensitivity of this method.

[0105] The detection results are as follows Figure 3 shown. It can be seen from the figure that the method of the present invention can detect 3.98 copies / μL of the real sample of Taura syndrome virus in the hepatopancreas of shrimp.

[0106] Example 5 Specificity Experiment

[0107] Select the cloned plasmids of the amplified target fragments of Taura syndrome virus, Yellow head virus, White spot syndrome virus, Infectious myonecrosis virus, Vibrio parahaemolyticus, Hepatopancreatic microsporidium, and Infectious myonecrosis virus synthesized by General Biosystems as templates for detection. At the same time, use the HPV positive plasmid as a positive control and no template as a negative control. Use the kit prepared in Example 2 and the detection method provided in Example 3 for detection (select a qPCR instrument for LAMP amplification).

[0108] The results are as follows Figure 4 shown. It can be seen from the figure that only the Taura syndrome virus in the hepatopancreas of shrimp shows positive amplification, and other results are all negative. The results indicate that the method of the present invention has no cross-reaction with the above-mentioned shrimp disease pathogens.

[0109] Example 6 Evaluation of Actual Samples

[0110] Collect 8 hepatopancreas tissues of actual sample shrimp, extract nucleic acids from the hepatopancreas tissues of shrimp, and use the Tiangen Marine Animal Tissue Genomic DNA Kit (product number DP324) as the extraction kit. Use the ordinary qPCR method, the kit prepared in Example 2, and the detection method provided in Example 3 for detection (select a thermostatic instrument for LAMP amplification).

[0111] Table 5

[0112] Sample number qPCR test result MB-LAMP test result 1 + + 2 - - 3 - - 4 - - 5 + + 6 + + 7 + + 8 + +

[0113] The detection results are shown in Table 5 and Figure 5 shown. It can be seen from this that the results of detecting the real sample of Taura syndrome virus in the hepatopancreas of shrimp by the method of the present invention using a thermostatic instrument are consistent with the qPCR results and also conform to the actual results.

[0114] Example 7 Stability Study

[0115] The kit prepared in Example 2 was stored at the actual storage condition (-20±5°C) for 11 months and then detected, and its performance was stable; the results of the accelerated stability experiment at 37°C showed that the kit was stored in a 37°C incubator for 24 hours, and the results met the quality requirements; the freeze-thaw stability test showed that at the actual storage temperature, after one freeze-thaw at each detection time point and continuous detection for 3 times, the results all met the quality requirements.

[0116] The above has described the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant art.

Claims

1. A primer-probe combination for detecting Taura syndrome virus, characterized in that, The primer-probe combination includes outer primer pairs F3 and B3, inner primer pairs FIP and BIP, and molecular beacon probe MB; The nucleic acid sequences of the outer primer pairs F3 and B3 are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively; The nucleic acid sequences of the inner primer pairs FIP and BIP are shown as SEQ ID NO: 3 and SEQ ID NO: 4 respectively; The nucleic acid sequence of the molecular beacon probe MB is shown as SEQ ID NO:

5.

2. The primer-probe combination according to claim 1, wherein The 5'-end of the molecular beacon probe MB is labeled with a fluorescent reporter group, and the 3'-end is labeled with a fluorescent quenching group; Preferably, the fluorescent reporter group includes one of FAM, TET, JOE, Cy3, Cy5, Cy5.5, Lluorescein, Rhodamine, Rhodamine Red, Rhodamine 6G, Orengon Green 488, Orengon Green 500, Orengon Green 514, Texas Red, TAMRA, Inosine, HEX, FITC, Acridine orange and ROX; Preferably, the fluorescent quenching group includes one of Dabcyl, DABSYL, TAMRA, BHQ-1, BHQ-2 and BHQ-3; And / or, the 9 bases at the 5'-end and the 9 bases at the 3'-end of the molecular beacon probe MB are completely complementary to form a stem-loop structure.

3. A kit, characterized in that, The kit contains the primer-probe combination as described in claim 1 or 2.

4. The kit according to claim 3, wherein The kit further includes at least one of a positive control, a negative control, and a LAMP reaction premix; Preferably, the LAMP reaction premix includes LAMP Buffer, Bst enzyme, and RNase HⅡ; More preferably, the LAMP Buffer comprises Tris-HCl, (NH4)2SO4, KCl, MgSO4, 20, dNTP; Preferably, the positive control includes a recombinant vector containing a nucleic acid molecule of Taura syndrome virus; Preferably, the negative control includes water.

5. Use of the primer combination according to any one of claims 1-2 or the kit according to any one of claims 3-4 in any of the following: 1) Detection or auxiliary detection of Taura syndrome virus for non-diagnostic purposes; 2) Preparation of a product for detecting or assisting in the detection of Taura syndrome virus.

6. The application according to claim 5, characterized in that, The product includes those selected from reagents, kits or chips.

7. The application according to claim 5, wherein The usage method of the product includes the following steps: Detecting the DNA of the sample to be tested using the primer combination according to any one of claims 1-2 or the kit according to any one of claims 3-4; Preferably, the detection includes using the DNA of the sample to be tested as a template, performing fluorescence loop-mediated isothermal amplification using the above primer-probe combination or kit, monitoring the fluorescence signal, and judging the result.

8. A method for detecting or assisting in the detection of Taura syndrome virus for non-diagnostic purposes, characterized in that, Including the following steps: Detecting the DNA of the sample to be tested using the primer combination according to any one of claims 1-2 or the kit according to any one of claims 3-4; Preferably, the detection includes using the DNA of the sample to be tested as a template, performing fluorescence loop-mediated isothermal amplification with the above primer-probe combination or kit, monitoring the fluorescence signal, and judging the result; Preferably, the reaction system for the fluorescence loop-mediated isothermal amplification includes:

9. The method according to claim 8, wherein The fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument or a constant temperature device; Preferably, the reaction system for the fluorescence loop-mediated isothermal amplification includes: Preferably, when the fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument, the amplification program is: 58 - 65 °C for 0.5 - 2 min, 40 - 48 cycles, and fluorescence data is collected; Preferably, when the fluorescence loop-mediated isothermal amplification is performed using a qPCR instrument, the result judgment is as follows: If a positive amplification curve is shown, the sample to be tested is positive and contains Taura syndrome virus; if a positive amplification curve is not shown, the sample to be tested is negative and does not contain Taura syndrome virus; Preferably, when the fluorescence loop-mediated isothermal amplification is performed using a constant temperature device, the amplification program is: 58 - 65 °C for 40 - 50 min; Preferably, when the fluorescence loop-mediated isothermal amplification is performed using a constant temperature device, when the fluorescence loop-mediated isothermal amplification is performed using a constant temperature device, the result judgment is as follows: The product obtained by the fluorescence loop-mediated isothermal amplification is irradiated under an ultraviolet lamp. If there is an obvious fluorescence signal, the sample to be tested is positive and contains Taura syndrome virus; if there is no fluorescence signal, the sample to be tested is negative and does not contain Taura syndrome virus.

10. A system for detecting Taura syndrome virus, characterized in that, Including: A detection module: used for performing fluorescence loop-mediated isothermal amplification on the nucleic acid of the sample to be tested using the primer combination according to any one of claims 1 - 2 or the kit according to any one of claims 3 - 4; An analysis module: used for evaluating whether the sample to be tested contains Taura syndrome virus.

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