Reagent and kit for detecting viral nervous necrosis virus of fish

Abstract

The invention discloses a reagent and a kit for detecting viral nervous necrosis virus of fish, and belongs to the technical field of virus detection. The reagent comprises a fish viral nervous necrosis virus upstream primer, a fish viral nervous necrosis virus downstream primer and a specific fluorescent probe, nucleic acid of a sample to be detected is subjected to real-time fluorescent quantitative RT-PCR amplification, and the sample to be detected is analyzed according to a real-time fluorescent quantitative amplification curve. The kit is high in specificity and detection sensitivity, the lowest detection limit can reach 100 copies / mu L, operation is easy, convenient and rapid, and the kit is suitable for field detection and has a wide application scene.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and particularly relates to a reagent and a kit for detecting viral nervous necrosis virus of fish. Background Art

[0002] Viral nervous necrosis virus (VNNV) is an RNA virus, and its genome consists of two molecules of single-stranded positive-strand RNA. It is the pathogen of viral nervous necrosis (VNN). At present, the number of hosts infected by VNNV has spread from marine fish to freshwater fish and invertebrates, with more than 120 host species, causing a major blow to the marine fish aquaculture industry. Currently, there is no effective treatment method for VNNV, and the key measure to reduce the harm of this disease is to focus on the purification and prevention of VNNV.

[0003] The existing detection methods for VNNV mainly include cell culture and identification, nested PCR, real-time fluorescence PCR, RT-LAMP, RT-RAA, colloidal gold test strip, ELISA, etc. Due to the large number of discoveries of VNNV strains and the continuous emergence of variant strains, the number of VNNV strains and RNA2 genes has also increased sharply, and the number of RNA2 genes has exceeded 1000; the large number of emerging target genes has affected various key indicators at the technical level of real-time RT-PCR: such as the selection of the target gene region, the conservation of the upstream and downstream primer sequences and positions. More seriously, the number of other aquatic RNA and DNA virus strains has also increased sharply; the number of virus host spectra has also increased sharply; these variables have affected the specificity, non-specificity and accuracy of the existing detection technologies. The existing detection technologies are difficult to cover the rapidly increasing strain gene target sequences, and the results lack accuracy and reliability. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a reagent and a kit for detecting viral nervous necrosis virus of fish, so as to solve the technical problem of poor detection sensitivity of the existing reagent.

[0005] To achieve the above object, the technical solution adopted by the present invention is: to provide a reagent for detecting viral nervous necrosis virus of fish, including an upstream primer, a downstream primer and a specific fluorescent probe for viral nervous necrosis virus of fish. The nucleotide sequence of the upstream primer for viral nervous necrosis virus of fish is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer for viral nervous necrosis virus of fish is shown in SEQ ID NO.2, and the nucleotide sequence of the specific fluorescent probe is shown in SEQ ID NO.3.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows:

[0007] Further, the 5'-end of the specific fluorescent probe is labeled with a fluorescent reporter group, and the 3'-end is labeled with a fluorescent quenching group.

[0008] Further, the fluorescent reporter group is FAM, and the fluorescent quenching group is BHQ1.

[0009] The present invention also discloses a kit comprising the above reagents for detecting viral nervous necrosis virus of fish.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows:

[0011] Further, the kit further includes a standard of viral nervous necrosis virus of fish.

[0012] Further, the standard of viral nervous necrosis virus of fish is a standard plasmid with a nucleotide sequence as shown in SEQ ID NO.4, and the standard plasmid contains a partial sequence of the conserved region gene of viral nervous necrosis virus of fish.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1. Simple operation: No special reagents are required, and no cumbersome steps such as pre-unwinding are needed. Only a constant-temperature fluorescence instrument is required, and the conditions are relatively mild. The whole reaction is simple and rapid. The amplification result can be directly judged according to the real-time fluorescence data, and no high-temperature cycle is required. Therefore, it is especially suitable for use in non-laboratory detection sites with a large number of samples, which is particularly urgent and necessary for inspection and quarantine as well as production practice.

[0015] 2. High sensitivity: The lowest detection limit of the present invention can reach 100 copies / μL.

[0016] 3. High specificity: The present invention does not amplify the plasmid DNA of nervous necrosis virus (VNNV), infectious pancreatic necrosis virus (IPNV), spring viremia of carp virus (SVCV), viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV) and koi herpesvirus disease (KHV), and has strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shows the influence of different primer concentrations (2.5, 5, 10, 20 and 30 μM) on the detection results;

[0018] Figure 2 Shows the influence of different primer concentrations (4, 6, 8, 10 and 12 μM) on the detection results;

[0019] Figure 3Effect of different probe concentrations (5, 10, 15, 20, and 25 μM) on the detection results;

[0020] Figure 4 Effect of different probe concentrations (6, 8, 10, 12, and 14 μM) on the detection results;

[0021] Figure 5 Effect of different annealing temperatures (55, 60, 65, 70, and 75 °C) on the detection results;

[0022] Figure 6 Effect of different annealing temperatures (56, 58, 60, 62, and 64 °C) on the detection results;

[0023] Figure 7 Amplification results of the standard product;

[0024] Figure 8 Standard curve for absolute quantification;

[0025] Figure 9 Fluorescent quantitative amplification curve of the detected sample;

[0026] Figure 10 Sensitivity detection results;

[0027] Figure 11 Specificity detection results. Detailed implementation manners

[0028] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art of this technology to understand the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0029] Example 1

[0030] 1. Primer design

[0031] Using the highly conserved sequence matched by fish nervous necrosis virus on NCBI (The National Center for Biotechnology Information / https: / / www.ncbi.nlm.nih.gov / ) as a template, a pair of specific primers (SEQ ID NO.1 and SEQ ID NO.2) and a detection probe (SEQ ID NO.3) were designed, and the primers and probe were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0032] 2. Synthesis of target sequence / standard plasmid

[0033] Based on the sequences of RGNNV AF5349983, AF550395, AF550394, MF144241, KY930894, Nc008041, the 403Nc sequence was selected in its RNA2 region; the sequence TAATACGACTCACTATAGGGtcacctg was added. It was synthesized by Shanghai Sangon Biotech Co., Ltd. and inserted into the pUC57 cloning plasmid.

[0034] The sequences of the specific primers, detection probe and standard plasmid are shown in Table 1.

[0035] Table 1 Primer sequence table

[0036]

[0037] Example 2

[0038] A detection method for fish viral nervous necrosis virus, comprising the following steps:

[0039] 1. Extraction of fish nervous necrosis virus RNA

[0040] (1) For RNA extraction, the Novizan RNA-easy Isolation Reagent kit (R701) was used: The ground fish head sample was transferred to a centrifuge tube, and 500 μL of RNA-easy (RNA-easy Isolation Reagent) was added to every 25 mg of tissue, and it was vigorously shaken or pipetted to fully lyse the sample.

[0041] (2) 2 / 5 volume of RNase-free ddH2O (200 μL for every 500 μL of RNA-easy) was added to the above lysate, and it was mixed by inverting up and down and allowed to stand at room temperature for 5 min.

[0042] (3) Centrifuge at 11200 rpm (12000×g) at room temperature for 15 min.

[0043] (4) Take out the centrifuge tube. At this time, the solution is divided into the upper aqueous phase (containing RNA) and the dark lower precipitate (containing impurities such as proteins, DNA, and polysaccharides). Aspirate the upper aqueous phase into a new centrifuge tube.

[0044] (5) Add an equal volume of isopropanol, invert the tube up and down to mix well, and let it stand at room temperature for 10 min. Centrifuge at 11200 rpm (12000×g) at room temperature for 10 min. A white precipitate can be seen. Discard the supernatant.

[0045] (6) Add 500 μL of 75% ethanol (prepared with RNase-free ddH2O), flick the bottom of the tube to suspend the precipitate, and invert the tube up and down several times.

[0046] (7) Centrifuge at 9100 rpm (8000×g) at room temperature for 3 min, and discard the supernatant.

[0047] (8) Repeat steps 6 and 7 once to discard all the supernatant.

[0048] (9) Let it dry at room temperature, add an appropriate amount of RNase-free ddH2O to dissolve the precipitate, and vortex at room temperature for 3 min to fully dissolve the RNA precipitate.

[0049] 2. Real-time fluorescence quantitative RT-PCR experiment

[0050] Use the Novoprotein kit III U+One Step qRT-PCR Probe Kit (Q225) for the experiment. The reaction system is shown in Table 2, and the reaction program is shown in Table 3.

[0051] Table 2 Reaction system

[0052]

[0053]

[0054] Table 3 Reaction program

[0055]

[0056] 3. Optimization of reaction conditions

[0057] (1) Primer concentration optimization

[0058] Set the primer concentrations to 2.5, 5, 10, 20, and 30 μM respectively, and set a negative control group to explore the effect of different primer concentrations on the amplification effect. The results are as Figure 1 shown. According to the experimental results obtained under different primer concentration conditions, the primer pair has good amplification performance within the concentration range of 10 - 20 μM, and the amplification performance increases limitedly above 20 μM.

[0059] Five primer concentrations (4, 6, 8, 10, and 12 μM) were further selected for the amplification reaction, and the results are as Figure 2 shown, and the optimal concentration of the primer was obtained as 10 μM.

[0060] (2) Probe concentration optimization

[0061] The probe concentrations were set to 5, 10, 15, 20, and 25 μM respectively, and a negative control group was set to explore the effect of different probe concentrations on the amplification effect. The results are as Figure 3 shown. According to the experimental results obtained under different probe concentration conditions, the probe had good amplification performance in the concentration range of 10 - 15 μM.

[0062] Five probe concentrations (6, 8, 10, 12, and 14 μM) were further refined and selected for the amplification reaction, and the results are as Figure 4 shown, and the optimal concentration of the probe was obtained as 8 μM.

[0063] (3) Annealing temperature optimization

[0064] Based on the optimal primer concentration and probe concentration, the annealing temperature was optimized. The annealing temperatures were set to 55, 60, 65, 70, and 75 °C respectively for PCR reaction, and the amplification curves are as Figure 5 shown. The annealing temperature had good amplification performance in the range of 55 - 60 °C.

[0065] Five annealing temperatures (56, 58, 60, 62, and 64 °C) were further refined and selected for the amplification reaction, and the results are as Figure 6 shown, and the optimal annealing temperature was obtained as 58 °C.

[0066] 4. Standard curve

[0067] Nine concentration gradients (4.0×10 10 , 4.0×10 9 , 4.0×10 8 , 4.0×10 7 , 4.0×10 6 , 4.0×10 5 , 4.0×10 4 , 4.0×10 3 and 4.0×10 2 copies / μL) of the standard product were used as the template RNA for RT-Real-time qPCR reaction, and the amplification results of the RNA standard product are as Figure 7 shown. Then, with the Ct value as the ordinate and the standard plasmid concentration as the abscissa, the graph is drawn as Figure 8The standard curve shown. The results show that the linear regression equation of the standard curve for detection using the primer-probe combination is Y = -3.232X + 40.921, and the correlation coefficient R 2 = 0.992 (>0.99), and the amplification efficiency is 103.886%.

[0068] 5. Sample Detection

[0069] Three Sebastes schlegelii were dissected, and their head kidneys and spleens were taken for total RNA extraction. Then, the above real-time fluorescence quantitative RT-PCR experiment was carried out using the extracted total RNA. The fluorescence quantitative amplification curves after RNA extraction are as Figure 9 shown, including the RNA of the head kidneys and spleens of three Sebastes schlegelii and negative controls, with three replicates each. Among them, VNNV amplification occurred in all three fish, and the CT value was around 27. In the negative control, the CT value was 35. Substituting into the standard curve ( Figure 8 ), the VNNV gene copy number of the sample fish was approximately 4×10 3 copies / μL.

[0070] Example 3 Sensitivity Detection

[0071] According to Figure 7 the amplification results of the 9 concentration gradients (4.0×10 10 , 4.0×10 9 , 4.0×10 8 , 4.0×10 7 , 4.0×10 6 , 4.0×10 5 , 4.0×10 4 , 4.0×10 3 and 4.0×10 2 copies / μL) of the RNA standard product were further divided into 5 concentration gradients (400, 200, 100, 50, and 25 copies / μL) for sensitivity experiments. The results are as Figure 10 shown. When the concentration was below 100 copies / μL, the curves could no longer be distinguished. It shows that this method has high sensitivity, and the lowest detection limit can reach 100 copies / μL.

[0072] Example 4 Repeatability Experiment

[0073] Using 3 concentration gradients (4.0×10 9 , 4.0×10 8 and 4.0×10 7Using the RNA standard product ([x] copies / μL) as a template, intra-group and inter-group repeatability tests were performed. The coefficient of variation of Ct values within and between groups is shown in Table 4. The test results showed that there was no significant difference in Ct values, indicating that the established method had good repeatability and stability.

[0074] Table 4 Results of Real-time qPCR Repeatability Assessment

[0075]

[0076] Example 5 Specificity Experiment

[0077] Using the method of the present invention, plasmid standards of nervous necrosis virus (VNNV), infectious pancreatic necrosis virus (IPNV), spring viraemia of carp virus (SVCV), viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV) and koi herpesvirus (KHV) (the plasmid standards were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.) were detected. The experimental results are as Figure 11 shown. It can be seen from the figure that the detection method specifically amplified only VNNV, and the other amplifications were not strong, indicating that its primers and probes had strong specificity.

Claims

1. A reagent for detecting viral nervous necrosis virus of fish, characterized in that, The reagent includes an upstream primer, a downstream primer and a specific fluorescent probe for viral nervous necrosis virus of fish. The nucleotide sequence of the upstream primer for viral nervous necrosis virus of fish is shown as SEQ ID NO.

1. The nucleotide sequence of the downstream primer for viral nervous necrosis virus of fish is shown as SEQ ID NO.

2. The nucleotide sequence of the specific fluorescent probe is shown as SEQ ID NO.

3.

2. The reagent for detecting viral nervous necrosis virus of fish according to claim 1, wherein The 5' end of the specific fluorescent probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group.

3. The reagent for detecting viral nervous necrosis virus of fish according to claim 2, wherein The fluorescent reporter group is FAM, and the fluorescent quenching group is BHQ1.

4. A kit for detecting viral nervous necrosis virus of fish, characterized in that, The kit contains the reagent for detecting viral nervous necrosis virus of fish according to any one of claims 1-3.

5. The kit for detecting viral nervous necrosis virus of fish according to claim 4, characterized in that, The kit further includes a standard for viral nervous necrosis virus of fish.

6. The kit for detecting viral nervous necrosis virus of fish according to claim 5, wherein The standard for viral nervous necrosis virus of fish is a standard plasmid with a nucleotide sequence shown as SEQ ID NO.4.

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