A kit and method for detecting blue crab reovirus in aquaculture water

Abstract

The present invention relates to the field of virus detection, and specifically to a kit and method for detecting blue crab reovirus in aquaculture water. Through experimental comparison, the present invention has found the best solution for enriching blue crab reovirus in water bodies, and combined with high-sensitivity fluorescent quantitative RT-PCR technology, established a detection method for blue crab reovirus in aquaculture water bodies. The present invention enriches blue crab reovirus in water bodies through conventional ultrafiltration tubes, significantly shortens the virus enrichment time, and improves the virus enrichment efficiency; on this basis, high-sensitivity fluorescent quantitative RT-PCR is used for virus detection, ensuring the sensitivity and accuracy of the detection. The present invention effectively fills the gap in the current method for detecting blue crab reovirus in water bodies, and has significant economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of virus detection, in particular to a kit and method for detecting blue crab reovirus in aquaculture water. Background Art

[0002] Mud Crab Reovirus (MCRV) is easily transmitted and prevalent, and is currently one of the most important pathogens in farmed mud crabs. Epidemiological studies in recent years have shown that the virus is present in almost all mud crab farming areas, posing a huge threat to the healthy development of the mud crab farming industry. The virus is a non-enveloped virus with strong tolerance to the natural environment. The virus is carried by water during the reproduction of mud crabs, making it easier for it to spread during seed production and farming. Our previous studies have found that MCRV can exist in aquaculture water for a long time, and conventional disinfectants have limited killing effects. Therefore, establishing a method for detecting MCRV in water bodies and conducting follow-up monitoring are of guiding significance for the production of MCRV-free seed and the healthy development of the mud crab farming industry.

[0003] Currently, insufficient attention has been paid to the issue of MCRV contamination in aquaculture water, and a corresponding monitoring system has yet to be established. Because the virus content in water is much lower than that in tissues, it is difficult to directly detect viruses in water samples. Enrichment and concentration of viruses in water samples are necessary as a prerequisite for subsequent pathogen detection. Viruses in water often exist in free or adsorbed forms on water particles. Filtration, polyethylene glycol precipitation, ultrafiltration tubes, high-speed centrifugation, and ultracentrifugation are some of the common methods for enriching water pathogens. Collecting MCRV-containing water samples for experimental and comparative analysis and selecting appropriate MCRV enrichment methods are crucial for effectively monitoring MCRV levels in water.

[0004] In addition, with the development of molecular biology technology, real-time fluorescence quantitative technology has been widely used in the detection of different types of viruses due to its many advantages such as high sensitivity in detecting target genes, strong reaction specificity, good repeatability and less operational pollution. Summary of the Invention

[0005] The present invention aims to provide a kit and method for detecting MCRV in aquaculture water. By fully enriching the virus and combining it with real-time fluorescence quantitative RT-PCR, a method for detecting MCRV in water during the aquaculture and breeding of blue crabs has been established. This technology can provide essential technical support for the prevention and control of MCRV, and even for the purification of blue crab seedlings from MCRV.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Through experimental comparison, the present invention has identified the optimal method for enriching MCRV in water bodies and, combined with highly sensitive fluorescent quantitative RT-PCR technology, established a method for detecting blue crab reovirus in aquaculture water bodies. This method enriches MCRV in water bodies using conventional ultrafiltration tubes, significantly shortening the virus enrichment time and improving the virus enrichment efficiency. On this basis, highly sensitive fluorescent quantitative RT-PCR is used for virus detection, ensuring the sensitivity and accuracy of the detection. This invention effectively fills the gap in current MCRV detection methods in water bodies and has significant economic and social benefits.

[0008] The first aspect of the present invention provides a detection kit for blue crab reovirus in aquaculture water, the kit comprising an ultrafiltration tube for concentrating water viruses and a highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent.

[0009] The ultrafiltration tubes are commercially available, conventional laboratory consumables, and different brands are expected to have similar filtration performance. For ease of operation, the present invention uses the most common Millipore 50kDa ultrafiltration centrifuge tubes, which can achieve a single sample loading volume of up to 15ml and a multiple loading volume of up to 50ml, with sample concentrations of approximately 300-500 times.

[0010] Furthermore, the ultrafiltration tube contains a cellulose membrane for retaining virus particles, and the theoretical molecular weight of the protein retained by the cellulose membrane of the ultrafiltration tube should be 50 kDa or greater than 50 kDa.

[0011] Furthermore, the minimum single sample loading volume of the ultrafiltration tube is 4 ml / tube, and water samples can be loaded continuously multiple times, and each tube can detect up to 50 ml of water at a time.

[0012] Furthermore, the ultrafiltration tube has a maximum speed of 4000 g / min using a hanging basket centrifuge or a maximum speed of 5000 g / min using an angle rotor, and the minimum volume of concentrated viruses is less than 100 μl.

[0013] Furthermore, the aquaculture water sample to be tested added to the ultrafiltration tube needs to be allowed to stand at room temperature for 1 hour in advance to precipitate large particles, and then filtered with medical gauze. The filtrate is centrifuged at 3000 r / min for 10 minutes to obtain the supernatant to remove algae and medium-sized suspended matter.

[0014] The kit of the present invention uses an ultrafiltration tube for virus concentration, with a theoretical virus recovery rate exceeding 95%. The entire virus concentration process takes no more than 3 hours, which is significantly superior to other common virus concentration methods such as ultracentrifugation, polyethylene glycol precipitation, high-speed centrifugation and ultrafiltration.

[0015] The present invention found through experimental comparison that under the condition of concentrating the same volume of water, most viruses will be retained when MCRV is concentrated using ultrafiltration tubes. The ultrafiltration tube method obtains the highest viral RNA concentration, and the virus content C T The lowest value (the lower the value, the higher the viral content); ultracentrifugation and polyethylene glycol precipitation yielded similar results, but ultracentrifugation was slightly better than polyethylene glycol precipitation. Direct high-speed centrifugation of water resulted in significantly greater viral loss. Furthermore, the ultrafiltration tube method significantly shortened the viral concentration time compared to ultracentrifugation and polyethylene glycol precipitation. Therefore, the ultrafiltration tube method offers significant advantages for MCRV concentration. Specific experimental results are shown in Table 1.

[0016] Furthermore, the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent includes a pair of specific primers designed based on the ORF region of viral genome segment 11 (VP11). The primer design refers to the MCRV genome VP11 segment sequence (GenBank No. HQ414137.1) and the VP11 segment sequences of two other isolated strains (SsRV, GenBank No. HQ414137.1; MCRV-NH, SEQ ID NO: 1). The coding region of the viral VP11 gene is predicted using online software, and a pair of quantitative primers are designed in the conserved region within the ORF region of the VP11 gene: the upstream primer VP11-F sequence is 5′–GTC AGA ATG TCGTTC ATA CTT TGT–3′ (SEQ ID NO: 2), and the downstream primer VP11-R sequence is 5′–ATT CAG GAG TTC CGGACA GAT–3′ (SEQ ID NO:3); and also includes a specific TaqMan probe based on VP11, the probe design is based on the conserved region of the VP11 gene, and the probe sequence VP11-Probe: 5′–FAM-CTG ATG CGT TCG ATT-MGB–3′ (SEQ ID NO:4).

[0017] Furthermore, the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent also includes a Taq enzyme premix reagent suitable for a fluorescent probe (for example, 2×Premix Ex Taq (Probe qPCR)), a reverse transcriptase premix reagent (for example, Primescript RT Master Mix, containing random primers Random6), a gradient dilution standard plasmid, a positive control, and a negative control (sterile double-distilled water). The Taq enzyme premix reagent and reverse transcription reagent contained in the reagent are both commercially available reagents, and 2×Premix Ex Taq (Probe qPCR)) and Primescript RT Master Mix are reagents recommended for use in the present invention, and both are available but not limited to these two reagents. The standard plasmid is a constructed double-stranded DNA vector containing the full length of the VP11 gene ORF region, which is pMD19T-VP11. The concentration range of the standard plasmid (1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 The positive control was a diluted standard plasmid (1×10 5 copies / μL), and the negative control was ddH2O.

[0018] Furthermore, the PCR reaction system for the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent is as follows: 10 μL of 2× Premix Ex Taq (Probe qPCR), 0.4 μL each of VP11-F and VP11-R (10 μM), 0.4 μL of VP11-Probe (10 μM), 0.4 μL of ROX (added as needed by the instrument), 2 μL of cDNA template, and 6.8 μL of ddH2O, for a total reaction volume of 20 μL. The addition of ROX reference dye depends on the instrument type; some instruments do not require the addition of reference dye.

[0019] Furthermore, the PCR reaction procedure of the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent is: pre-denaturation at 95°C for 30s; then denaturation at 95°C for 5s, annealing and extension at 60°C for 30s, and 40 cycles.

[0020] Furthermore, the method for detecting viruses using the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent comprises the following steps:

[0021] (a) Total RNA was extracted from water and RNA quality was tested; DNase I was added to RNA samples of qualified quality to remove DNA contamination;

[0022] (b) During reverse transcription, in addition to reverse transcriptase and RNase inhibitor, the reverse transcription system must also include random primer Random6 to ensure reverse transcription efficiency and be placed in a 42°C water bath for 30 min.

[0023] (c) In the prepared fluorescent quantitative RT-PCR reaction system, in addition to containing 10 μL of 2× Premix Ex Taq (ProbeqPCR), 0.4 μL each of VP11-F and VP11-R (10 μM), 0.4 μL of VP11-Probe (10 μM), 0.4 μL of ROX (optional), 2 μL of DNA template (sample nucleic acid to be tested, positive or negative control sample), and 6.8 μL of ddH2O, the total reaction volume is 20 μL;

[0024] (d) The reaction conditions of fluorescence quantitative RT-PCR were as follows: pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s and annealing and extension at 60°C for 30 s for 40 cycles.

[0025] (e) Determine the presence of MCRV based on the Ct value generated by the fluorescent quantitative RT-PCR reaction and calculate the virus content in the water.

[0026] A second aspect of the present invention provides a method for detecting blue crab reovirus in aquaculture water using the above-mentioned kit, comprising a virus concentration process and a pathogen detection process, specifically comprising the following steps:

[0027] (A) The aquaculture water sample to be tested must be allowed to stand at room temperature for 1 hour to allow large particles to settle. The sample is then filtered through medical gauze and the filtrate is centrifuged at 3000 rpm for 10 minutes to obtain the supernatant.

[0028] (B) Repeatedly add the supernatant (centrifuge each tube multiple times and add continuously), with the total sample volume not exceeding 50 ml.

[0029] (C) placing the ultrafiltration tube in a hanging basket and centrifuging at a centrifugal force of 4000 g / min, or centrifuging at a speed of 5000 g / min using an angle rotor, and stopping the centrifugation when the volume of the concentrate is approximately 100 μl;

[0030] (D) Use a pipette to resuspend the virus concentrate and transfer it to a 2 ml centrifuge tube;

[0031] (E) Extract and concentrate viral RNA according to the instructions of the high-sensitivity fluorescent quantitative RT-PCR virus quantitative detection reagent, test the RNA quality, and add DNase I to the RNA sample of qualified quality to remove DNA contamination;

[0032] (F) During reverse transcription, in addition to reverse transcriptase and RNase inhibitor, the reverse transcription system also required the addition of random primer Random6 to ensure reverse transcription efficiency and was placed in a 42°C water bath for 30 min.

[0033] (G) The prepared fluorescent quantitative RT-PCR reaction system contains 10 μL of 2× Premix Ex Taq (Probe qPCR), 0.4 μL of primers VP11-F and VP11-R (10 μM), 0.4 μL of probe VP11-Probe (10 μM), 0.4 μL of ROX (optional), 2 μL of cDNA template (sample nucleic acid to be tested, positive or negative control sample), and 6.8 μL of ddH2O, for a total reaction volume of 20 μL.

[0034] (H) The reaction conditions of fluorescence quantitative RT-PCR were as follows: pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for 40 cycles.

[0035] (I) C produced by fluorescence quantitative RT-PCR reaction T value to determine whether there is viral infection and calculate the virus content in the water.

[0036] The advantages of the present invention are:

[0037] The present invention improves the practicality and sensitivity of the detection kit from two aspects: improving the virus concentration efficiency and adopting highly sensitive fluorescent quantitative RT-PCR:

[0038] 1. The ultrafiltration tube method is used for virus concentration. Compared with other conventional concentration methods, this method has the characteristics of high virus recovery rate, short time and low cost. It is a convenient and efficient method for water virus concentration.

[0039] (1) High virus recovery rate. By comparing several conventional water virus concentration methods, it was found that ultrafiltration tube concentration obtained the highest viral RNA concentration, indicating that the virus enrichment effect was the best. This is related to the characteristics of the ultrafiltration tube method itself. The ultrafiltration tube method of virus concentration is actually to intercept macromolecular substances through the pore size of the ultrafiltration membrane. Virus particles are much larger than 50kDa. In theory, all viruses in the water will be trapped in the ultrafiltration tube, ensuring high efficiency of virus recovery.

[0040] (2) Short virus concentration time. Through comparative experiments, we can find that under the premise of ensuring virus recovery efficiency, the ultrafiltration tube method takes the shortest time to concentrate the virus. Although the high-speed centrifugation method and the suction filtration method take shorter time, the high-speed centrifugation method causes greater virus loss, and the suction filtration method cannot obtain MCRV.

[0041] (3) The cost of virus concentration is lower. During the development of the present invention, comparative experiments showed that the cost of using the ultrafiltration tube method is significantly lower than that of the ultracentrifugation method. Although ultracentrifugation is a classic method for virus concentration, ultracentrifuge equipment is expensive and belongs to expensive experimental equipment. Its consumables are also expensive. In comparison, the ultrafiltration tube method only requires an ordinary centrifuge and ultrafiltration tubes to complete virus concentration.

[0042] 2. Highly sensitive fluorescent quantitative RT-PCR is used for virus detection to ensure the sensitivity and specificity of the detection.

[0043] (1) The detection method has the advantage of high expression levels of target genes. Blue crab reovirus expresses a total of 13 protein genes. Through expression level analysis, we found that the VP11 gene expression level was the highest. Most of the existing blue crab reovirus detection methods are based on the design of primers for the VP1 or VP6 genes. The expression levels of these two genes are significantly lower than that of VP11. In the same test sample, the design of primers based on highly expressed genes for pathogen detection is equivalent to a larger number of viral templates, which makes it easier to detect the presence of the virus and improves the sensitivity of the test.

[0044] (2) The fluorescence quantitative PCR detection technology of the detection kit itself is highly sensitive. Compared with conventional RT-PCR, nested RT-PCR, and colloidal gold technology, fluorescence quantitative PCR detection technology is more sensitive and is currently recognized and commonly used high-sensitivity pathogen detection technology. The lower limit of pathogen quantification of the MCRV detection technology established in this invention is 10 copies / reaction, and the lower limit of pathogen detection is 2.5 copies / reaction, which has reached the limit level of fluorescence quantitative PCR detection technology.

[0045] (3) The detection method has high sensitivity and specificity. When the detection method of the present invention was used to detect common pathogens of blue crabs and shrimp, it was found that no visible amplification curves were found in samples containing nucleic acids of MCDV, WSSV, DIV1, EHP, and Vibrio parahaemolyticus. This shows that the detection method has high specificity and the mixed infection or presence of these pathogens will not affect the MCRV detection results.

[0046] Therefore, the MCRV detection kit and detection method in water bodies established by the present invention based on the above two points have obvious comparative advantages and are the best feasible solution for detecting MCRV content in water bodies.

[0047] 3. The present invention provides a detection kit and method for detecting MCRV in blue crab reovirus in aquaculture water, which has great application value in the detection of MCRV in blue crab seed breeding water, aquaculture water, and water environment.

[0048] First of all, this detection method has a good application prospect in the detection of MCRV in water bodies during the breeding process of blue crab seedlings. MCRV is a conditional pathogen. When MCRV is contained in the water body, especially when the virus content is high, it is easy for the fertilized eggs and larvae of blue crabs to be infected with the pathogen. Since the virus content in the seedling water body is very low, the MCRV in the water body needs to be concentrated first, and with the help of a high-sensitivity detection method, false negatives can be avoided. The present invention has found the optimal concentration scheme for MCRV in water bodies, and uses the TaqMan high-sensitivity probe for pathogen detection, which can well meet the needs of pathogen detection in water bodies during the seedling breeding stage.

[0049] Secondly, this virus detection method also has promising application prospects in pathogen investigations in aquaculture water. Detecting MCRV in aquaculture water is a fundamental requirement for comprehensive epidemiological studies. Compared to seedling rearing water, aquaculture water has lower transparency and contains more suspended particles, such as algae. This method removes large impurities through static standing, filtration, and low-speed centrifugation, followed by virus concentration. This ensures the universality of this water virus detection method and meets the needs of aquaculture water virus detection.

[0050] Finally, this detection method can also be used to detect MCRV in environmental water bodies. Currently, with the development of green aquaculture, the requirements for water environment are also higher. The water MCRV detection method established by the present invention can also be used to investigate the MCRV carrying situation in the environment. According to our previous research, MCRV has a very strong ability to tolerate the environment and can survive for up to 3 months or even longer in the natural environment. MCRV carried from aquaculture water bodies into the larger water environment can easily cause environmental pollution and even pose a potential infection threat to other species. Therefore, this detection method also has potential application value in the detection of viruses in water environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 .MCRV standard curve; the horizontal axis is the copy number of the sample, the vertical axis is C T value.

[0052] Figure 2 . qRT-PCR amplification curve of standard plasmids; the concentrations of standard plasmids No. 1 to No. 8 in the figure are 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 and 1×10 1 copies / μL.

[0053] Figure 3. Sensitivity test; qRT-PCR amplification curve: the concentrations of plasmids No. 1 to No. 4 were 1×10 1 , 0.5×10 1 , 0.25×10 1 and 1×10 0 copies / μL.

[0054] Figure 4 Specificity detection; 1, positive control; 2-3, MCRV-positive samples; 4, MCDV; 5, WSSV; 6, DIV1; 7, EHP; 8, Vibrio parahaemolyticus; 9, negative control. DETAILED DESCRIPTION

[0055] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0056] Example 1: Ultracentrifugation can effectively enrich viruses

[0057] Ultracentrifugation is the most classic method for purifying and concentrating viruses. It uses the strong centrifugal force of the ultracentrifuge to centrifuge the viruses in the water to the bottom of the tube, thereby achieving the purpose of concentrating and purifying the viruses. The following steps are taken to concentrate viruses in water using an ultracentrifuge:

[0058] (a) Water pretreatment: The aquaculture water sample to be tested must be allowed to stand at room temperature for 1 hour to allow large particles to settle. The sample is then filtered through medical gauze and the filtrate is centrifuged at 3000 rpm for 10 minutes to obtain the supernatant.

[0059] (b) Ultracentrifugation: 50 mL of the treated water sample was transferred into a Beckman ultracentrifuge tube and then ultracentrifuged at 40,000 × g for 2 h at 4°C.

[0060] (c) Virus collection and lysis: After ultracentrifugation, remove as much supernatant as possible, add 1 mL of RNA lysis buffer, shake and mix, and lyse for 5 min;

[0061] (e) RNA extraction: Extract and concentrate viral RNA according to the kit instructions (e.g., Transzol UP Plus RNA Kit from Quanshijin). Test the RNA quality. For RNA samples that meet the quality requirements, add DNase I to remove DNA contamination.

[0062] (f) Reverse transcription: After the extracted total RNA is inspected and tested to meet the requirements, reverse transcription is performed. The specific steps of reverse transcription are carried out according to the operating instructions of the reverse transcription kit (for example, TAKARA Primescript RT Master Mix). During reverse transcription, in addition to reverse transcriptase, RNase inhibitor and RNA template, the reverse transcription system needs to add random primer Random6 to ensure the reverse transcription effect, and is placed in a 42°C water bath for 30 minutes. The synthesized cDNA is used for subsequent PCR amplification;

[0063] (g) Quantitative RT-PCR: quantitative primers (VP11-F: 5′–GTC AGA ATG TCG TTC ATA CTT TGT–3′, SEQ ID NO: 2); VP11-R: 5′–ATT CAG GAG TTC CGG ACA GAT–3′, SEQ ID NO: 3) and a TaqMan probe (VP11-Probe: 5′–FAM-CTG ATG CGT TCG ATT-MGB–3′, SEQ ID NO: 4). The prepared fluorescent quantitative RT-PCR reaction system contains 10 μL of 2× Premix Ex Taq (Probe qPCR), 0.4 μL each of primers VP11-F and VP11-R (10 μM), 0.4 μL of probe VP11-Probe (10 μM), 0.4 μL of ROX (optional), 2 μL of cDNA template (sample nucleic acid to be tested, positive or negative control sample), and 6.8 μL of ddH2O. The total reaction volume is 20 μL.

[0064] (h) The reaction conditions of fluorescence quantitative RT-PCR were as follows: pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for 40 cycles.

[0065] (i) Calculation of viral content: Based on the C T The MCRV content in the water can be calculated based on the standard plasmid.

[0066] The experimental results are shown in Table 1. Using 50 ml of pretreated water, the RNA concentration of the virus sample extracted by ultracentrifugation was 74.8 ng / μL. By quantitative RT-PCR analysis, the C T The value is 34.61.

[0067] Example 2: High-speed centrifugation has limited effect on virus enrichment

[0068] High-speed centrifugation uses the centrifugal force of an ordinary high-speed centrifuge to centrifuge suspended viruses or small particles adsorbed with viruses in a water sample to the bottom of a centrifuge tube. After discarding the supernatant, the settled virus particles and particles carrying virus particles are obtained at the bottom of the centrifuge tube. Compared with ultracentrifugation, high-speed centrifuges are conventional laboratory equipment and are easy to use, but the centrifugal force is relatively low, especially when the volume is large. The steps for concentrating viruses by high-speed centrifugation are as follows:

[0069] (a) Water pretreatment: refer to step (a) of Example 1;

[0070] (b) High-speed centrifugation: 50 mL of treated water was added to a 50 mL centrifuge tube and centrifuged at 10,000 × g for 20 min at 4°C.

[0071] (c) Virus collection and lysis: After high-speed centrifugation, remove as much supernatant as possible from the tube, add 1 mL of RNA lysis buffer, shake to mix, and lyse for 5 min;

[0072] (d) RNA extraction and fluorescence quantitative RT-PCR were carried out as described in step (di) of Example 1.

[0073] The experimental results are shown in Table 1. Using 50 ml of pretreated water, the RNA concentration of the virus sample extracted by high-speed centrifugation was 60.2 ng / μL. By quantitative RT-PCR analysis, the C T The value is 35.2.

[0074] Example 3: MCRV enrichment cannot be performed by filtration

[0075] Water sample filtration is the simplest concentration method. It mainly uses a vacuum pump to change the internal and external pressures. The water passes through the filter membrane under the action of the pressure difference, while the virus or adsorbed virus particles are trapped on the filter membrane. A large volume of water can be filtered at a time. The specific operation steps are as follows:

[0076] (a) Water pretreatment: refer to step (a) of Example 1;

[0077] (b) Vacuum filtration: Use a 0.22 μm filter membrane and add 100 mL of the treated water to a vacuum filtration pump for filtration.

[0078] (c) Virus collection and lysis: The filter membrane was chopped and immersed in 2 ml of RNA lysis buffer. After lysis for 5 minutes, the membrane was centrifuged at 10,000 rpm to separate the shredded filter membrane from the lysis buffer.

[0079] (d) RNA extraction and fluorescence quantitative RT-PCR operations were performed with reference to step (di) of Example 1.

[0080] The experimental results are shown in Table 1: The concentration of the virus RNA extracted from each 50 ml of pre-treated water was 0. When 1 μL of cDNA was used as a template, no S-shaped amplification curve appeared. T The value is Not Detected.

[0081] Example 4: Polyethylene glycol precipitation is a good enrichment method, but takes the longest time

[0082] Polyethylene glycol precipitation is a common method for enriching water viruses. The specific operation process for enriching MCRV is as follows:

[0083] (a) Water pretreatment: refer to step (a) of Example 1;

[0084] (b) Polyethylene glycol precipitation: 50 ml of pretreated aquaculture water was added with PEG8000 to a final concentration of 8%, and NaCl was added to a final concentration of 0.3 mol / L. The mixture was stirred and allowed to stand overnight at 4°C. The mixture was centrifuged at 10,000 rpm for 30 min in a high-speed refrigerated centrifuge and the supernatant was discarded to obtain the resuspended virus concentrate.

[0085] (c) Virus collection and lysis: Resuspend the viral particle pellet in 0.1 ml of PBS (pH 7.4), add 1 ml of RNA lysis buffer, shake to mix, and lyse for 5 min.

[0086] (d) RNA extraction and fluorescence quantitative RT-PCR operations were performed with reference to step (di) of Example 1.

[0087] The experimental results are shown in Table 1. The virus samples obtained by polyethylene glycol method for each 50 ml of pretreated water had an average RNA concentration of 73.3. The C T The value is 34.9.

[0088] Example 5: Ultrafiltration tube method is effective in concentrating water viruses

[0089] Ultrafiltration tubes are commonly used for protein concentration and are available in different sizes based on the molecular weight of the protein retained by the membrane and the pore size. Under the centrifugal force of the liquid in the ultrafiltration tube, small molecules can pass through the membrane while large molecules cannot, which can help concentrate virus particles in the water. We will use the following steps to concentrate and detect viruses in water samples:

[0090] (a) The aquaculture water sample to be tested must be allowed to stand at room temperature for 1 hour to allow large particles to settle. The sample is then filtered through medical gauze and the filtrate is centrifuged at 3000 rpm for 10 minutes to obtain the supernatant.

[0091] (b) The supernatant water was added to the sample several times continuously (each tube was centrifuged several times and added continuously), and the total volume of the sample did not exceed 50 ml;

[0092] (c) placing the ultrafiltration tube in a hanging basket and centrifuging at a centrifugal force of 4000 × g / min, or centrifuging at a speed of 5000 × g / min using an angle rotor, and stopping the centrifugation when the volume of the concentrate is approximately 100 μl;

[0093] (d) Resuspend the virus concentrate with a pipette, then invert the ultrafiltration tube into a 50 mL centrifuge tube, centrifuge the concentrate, transfer the concentrate to a 1.5 mL centrifuge tube, and add 1 mL of cell lysis buffer;

[0094] (e) RNA extraction and fluorescence quantitative RT-PCR operations were performed with reference to step (di) of Example 1.

[0095] The experimental results showed that the average concentration of RNA extracted from 50 ml of pre-treated water obtained by ultrafiltration was 80.3. The C T The value is 33.5.

[0096] In conclusion, compared with the above four concentration methods, the ultrafiltration tube method has the highest RNA concentration and the highest calculated viral content (C T smallest, indicating the highest viral load).

[0097] Table 1 Analysis of the effects of several common water virus enrichment methods

[0098]

[0099] Example 6: Calculation of primer amplification efficiency for qRT-PCR detection method based on TaqMan probe

[0100] (1) RNA extraction and reverse transcription

[0101] The cDNA synthesized according to the steps in Example 1 was used for quantitative analysis.

[0102] (2) Primers and probes

[0103] Refer to the primers (VP11-F, VP11-R) and probe (VP11-Probe) used in the fluorescent quantitative RT-PCR described in Example 1.

[0104] (3) TaqMan probe qRT-PCR reaction

[0105] In the prepared fluorescent quantitative RT-PCR reaction system, in addition to 2×Premix Ex Taq (Probe qPCR), ROX, forward primer VP11-F, reverse primer VP11-R and probe VP11-Probe, standard plasmids of different concentration gradients were added and sterilized double-distilled water was added to the total volume of 20μl. A preliminary experiment was first performed using the recommended standard procedure. The specificity and feasibility of the primers were determined by analyzing the melting curve of the qRT-PCR product. On this basis, the optimal ratio of the upstream and downstream primers (to obtain the minimum C T By changing the PCR reaction temperature, the optimal annealing temperature and reaction time were found, and the reaction parameters were finally determined.

[0106] (4) Establishment of qRT-PCR reaction system and conditions

[0107] The experimental results showed that when the final concentration of primers was 2 μmol / L and the annealing and reaction temperature was 60°C, the detection samples could obtain smaller C T The results showed that the PCR product had a higher fluorescence signal intensity and a higher fluorescence value. Screening experiments with primer and probe concentrations determined the optimal total reaction volume to be 20 μL: 10 μL of 2× Premix Ex Taq (Probe qPCR), 0.4 μL each of 10 μM VP11-F and VP11-R, 0.4 μL of 10 μM VP11-Probe, 0.4 μL of ROX, 2 μL of DNA template, and 6.8 μL of ddH2O. The optimized TaqMan qRT-PCR reaction protocol included pre-denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds.

[0108] (5) Calculation of qRT-PCR amplification efficiency

[0109] The standard sample stored in the laboratory (1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 100 copies / μL) as the reaction template, 5 replicates were set for each gradient, and the fluorescence quantitative PCR reaction was performed according to the method established in Example 3. The instrument automatically drew the standard curve and generated the linear equation and correlation coefficient R 2 The amplification efficiency of the primers was calculated based on the slope of the linear equation, and the C values ​​of each gradient were calculated using statistical methods. T The coefficient of variation (CV) of the values ​​was used to analyze their repeatability and stability. Figure 1 Shown: Linear equation: C T =-3.242X+40.442 (where X is the logarithm of the number of plasmids), the correlation coefficient of the linear equation is R 2 The slope of the standard curve was 0.998, indicating that the point values ​​on the standard curve had a good linear relationship, and the primer amplification efficiency calculated based on the slope of the standard curve was 103.416%, which was within the optimal range of this type of reaction (95-105%). T The coefficient of variation (CV) of the values ​​was between 0.2% and 1.4%, indicating that the detection method had good stability and repeatability (Table 2).

[0110] Table 2. C of MCRV standard plasmid T Value and coefficient of variation

[0111]

[0112]

[0113] Example 7: Detection method with high sensitivity

[0114] Based on the 10-fold serial dilution of the aforementioned standard plasmid, 10, 5, 2.5, and 1 copy / μL standard plasmids were further prepared. The serially diluted plasmids were used as templates for fluorescence quantitative PCR reaction. T The coefficient of variation of the value was used to determine the detection limit of the method; the corresponding C T Value, judge whether the virus infection degree and content. Figure 2 It can be seen that when the standard plasmid is used as a template, the plasmid numbers of 10, 5 and 2.5 copies / reaction can all be effectively amplified, and a good "S"-shaped amplification curve is obtained. When the plasmid concentration is 10 copies / μL, the repeatability between samples is very good, and the detection value is still within the linear range of the standard curve, indicating that this detection method can be used for the absolute quantification of pure virus particles with a sensitivity of up to 10 copies / μL. When the plasmid concentration is 5 or 2.5 copies / μL, the detection value is no longer linear and is no longer suitable for absolute quantification of the sample. Despite this, both gradient plasmid samples can obtain an "S"-shaped amplification curve, indicating that it can still be used for qualitative detection of viruses with a sensitivity of 2.5 copies / μL ( Figure 3 ).

[0115] Example 8: Detection method with strong specificity

[0116] To analyze the specificity of the detection method, samples of gill tissue carrying MCRV were used as positive controls, and cDNA samples of gill tissue of healthy blue crabs were used as negative controls. Fluorescence quantitative PCR reactions were performed using nucleic acid samples of common crustacean pathogens, such as MCDV (Mud Crab Dicistrovirus) cDNA and WSSV (White Spot Syndrome Virus), DIV1 (Decapod Iridescent Virus 1), EHP (Enterocytozoon hepatopenaei) or DNA of Vibrio parahaemolyticus. The experimental results showed that no visible amplification curves were observed when using samples containing nucleic acids of MCDV, WSSV, DIV1, EHP and Vibrio parahaemolyticus, indicating that the detection method has high specificity and will not affect the MCRV detection results due to mixed infection or presence of these pathogens ( Figure 4 ).

[0117] Example 9: Application of the detection kit to MCRV detection in water bodies of blue crab seedling farms

[0118] To further verify the practicality of the detection method and understand the MCRV contamination status of water bodies during the blue crab seedling breeding stage, the virus detection method established in Example 5 was used to track the water bodies collected from a blue crab seedling farm in Ninghai, Zhejiang, including the reservoir, water intake, seawater outside the sand filter, sand filter, dark sedimentation tank and seedling pond, and tap water was taken as a negative control. The test results are shown in Table 3: After MCRV infection occurred in the seedling farm, MCRV detection was performed on water samples from the bay water intake, outside the sand filter, sand filter, dark sedimentation tank, reservoir and seedling pond, respectively. The results are shown in Table 3. No MCRV was detected in the seawater intake, reservoir No. 1, or tap water, but MCRV was detected in reservoir No. 2, sand filter, dark sedimentation tank and seedling pond, indicating that virus contamination existed from reservoir No. 2 to the seedling pond. Considering that the virus content in reservoir No. 2 was the highest, it is likely that reservoir No. 2 was first contaminated by the virus, which eventually led to virus contamination in the seedling workshop.

[0119] Table 3 Survey on MCRV carrying in water bodies of blue crab seedling farms

[0120]

[0121] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application. Sequence Listing <110> East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences <120> A kit and method for detecting blue crab reovirus in aquaculture water <130> / <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 612 <212> DNA <213> Mud Crab Reovirus <400> 1 atgaataggt caaaagcaat aaacttccaa ccttttatgt tagaaactcg gccacccccta 60 accaccatcc ctataatgga ccagttggtt gaaattggag aacgttctaa tcaaaagtgg 120 agcatgaccg accggttgtt ctttgcgatt aggaagatca atcctatatt cgtcacttcg 180 agccagatac cttcaaaatt tgattacacc attctccaga tgcccactca gctaattgcc 240 tcattgaaag agacactttt gttcttagcc ttctcatatt acctaagaga atatcaagat 300 aaggttggtc aaatgaaatt ttacccagta gccatgaaaa acatgattcc tattgtcaac 360 tatctcaaag atcgtgttca taacaacttt gacactactt tggaacaggc atatcgtcag 420 aatgtcgttc atactttgtt tgcttctgat gcgttcgatt tactttccgg catgatcgct 480 actactagac ttgatctgat tcagaggacc aggatctgtc cggaactcct gaatgtactt 540 aacaaaatgt cctttatattct catttatgca ccaaatcgac catctatact ctcttggaaa 600 aaccaaagtt ga 612 <210> 2 <211> twenty four <212> DNA <213> Artificial <400> 2 gtcagaatgt cgttcatact ttgt 24 <210> 3 <211> twenty one <212> DNA <213> Artificial <400> 3 attcaggagt tccggacaga t 21 <210> 4 <211> 15 <212> DNA <213> Artificial <400> 4 ctgatgcgtt cgatt 15

Claims

1. A kit for detecting blue crab reovirus in aquaculture water, characterized in that: The kit comprises an ultrafiltration tube for concentrating water viruses and a highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent; the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent comprises a pair of specific primers, with the upstream and downstream primer sequences being shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and a specific TaqMan probe, with the probe sequence being shown in SEQ ID NO: 4; the ultrafiltration tube contains a cellulose membrane, and the theoretical molecular weight of proteins retained by the cellulose membrane of the ultrafiltration tube is 50 kDa or greater; the ultrafiltration tube has a maximum centrifugal speed of 4000 g / min using a hanging basket or a maximum centrifugal speed of 5000 g / min using an angle rotor, and the minimum volume of concentrated viruses is less than 100 μL.

2. The detection kit for blue crab reovirus in aquaculture water according to claim 1, characterized in that The highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent also includes a Taq enzyme premix reagent suitable for a fluorescent probe and a reverse transcriptase premix reagent containing a random primer Random6, a gradient dilution standard plasmid, a positive control, and a negative control sterilized double-distilled water.

3. The detection kit for blue crab reovirus in aquaculture water according to claim 2, characterized in that The PCR reaction system of the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent is: Probe qPCR 2×PremixEx Taq 10 μL, 0.4 μL each of 10 μM VP11-F and VP11-R, 0.4 μL of 10 μM VP11-Probe, 2 μL of cDNA template, 6.8 μL of ddH2O, and the total reaction volume is 20 μL.

4. The detection kit for blue crab reovirus in aquaculture water according to claim 3, characterized in that: The PCR reaction procedure of the highly sensitive fluorescent quantitative RT-PCR virus quantitative detection reagent is: pre-denaturation at 95°C for 30 seconds; then denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds, and 40 cycles.

5. A method for detecting blue crab reovirus in aquaculture water using the kit according to any one of claims 1 to 4, characterized in that: The process includes virus concentration and pathogen detection, specifically including the following steps: (A) The aquaculture water sample to be tested must be allowed to stand at room temperature for 1 hour to allow large particles to settle. The sample is then filtered through medical gauze and the filtrate is centrifuged at 3000 rpm for 10 minutes to obtain the supernatant. (B) The supernatant was loaded multiple times, with each tube centrifuged multiple times and added continuously, with the total loading volume not exceeding 50 mL; (C) placing the ultrafiltration tube in a hanging basket and centrifuging at a centrifugal force of 4000 g / min, or centrifuging at a speed of 5000 g / min using an angle rotor, and stopping the centrifugation when the volume of the concentrate is approximately 100 μL; (D) Use a pipette to resuspend the virus concentrate and transfer it to a 2 mL centrifuge tube; (E) Extract and concentrate viral RNA according to the instructions of the high-sensitivity fluorescent quantitative RT-PCR virus quantitative detection reagent, test the RNA quality, and add DNase I to the RNA sample of qualified quality to remove DNA contamination; (F) During reverse transcription, in addition to reverse transcriptase and RNase inhibitor, the reverse transcription system also required the addition of random primer Random6 to ensure reverse transcription efficiency and was placed in a 42°C water bath for 30 min. (G) The prepared fluorescent quantitative RT-PCR reaction system contains 10 μL of Probe qPCR 2× Premix Ex Taq, 0.4 μL of 10 μM primers VP11-F and VP11-R, 0.4 μL of 10 μM probe VP11-Probe, 2 μL of the sample nucleic acid to be tested, a positive or negative control sample, and 6.8 μL of ddH2O, for a total reaction volume of 20 μL. (H) Fluorescence quantitative RT-PCR reaction conditions were as follows: pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for 40 cycles; (I) C produced by fluorescence quantitative RT-PCR reaction T value to determine whether there is viral infection and calculate the virus content in the water.

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