Macrobrachium rosenbergii circular DNA virus and detection primer and kit thereof

Abstract

The invention discloses a macrobrachium rosenbergii circular DNA (deoxyribonucleic acid) virus as well as a detection primer and a kit thereof, the macrobrachium rosenbergii circular DNA virus is classified and named as macrobrachium rosenbergii circular DNA virus, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO: 46279, and the preservation date is December 19, 2024. The virus is closely related to the slow growth symptom of the macrobrachium rosenbergii, a research basis is provided for prevention and treatment of the iron shrimp syndrome of the macrobrachium rosenbergii, and meanwhile, a developed virus detection related technology fills up the blank that a nucleic acid molecule detection method for inducing the macrobrachium rosenbergii circular DNA virus does not exist at present; the kit is suitable for definite diagnosis, screening and prevention of the macrobrachium rosenbergii circular DNA virus.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a Macrobrachium rosenbergii circular DNA virus and a detection primer and a kit thereof. Background Art

[0002] Macrobrachium rosenbergii, also known as the freshwater giant prawn, has become one of the main freshwater aquaculture species in my country due to its excellent biological characteristics and good breeding benefits. According to surveys, my country's annual breeding of Macrobrachium rosenbergii reached about 28.6 billion in 2022, and the total production of Macrobrachium rosenbergii in 2023 reached 196,374 tons, making it the country with the largest production of Macrobrachium rosenbergii in the world.

[0003] However, highly intensive farming is accompanied by more challenges. Macrobrachium rosenbergii will have different diseases at different growth stages, including: larval mortality syndrome, larval metamorphosis syndrome, pathogenic enterobacteriaceae, ciliates, bell worms, etc. in the larval stage; white tail disease in the juvenile stage; white spot disease, gill disease, muscle necrosis, slow growth syndrome, white body vesicular disease, star disease and drag net syndrome in the grow-out stage. Among them, slow growth syndrome has appeared in Macrobrachium rosenbergii farming since 2010. Its main characteristics are precocious puberty and slow growth. It is also called iron prawn syndrome (IPS). "Iron prawn syndrome" is a description by farmers of the symptoms of early sexual maturity, hard shell and small growth in Macrobrachium rosenbergii farming. It is generally manifested as the interval between molting of Macrobrachium rosenbergii when it grows to 4-6 cm, and the growth slows down significantly. When it grows to 6-7 cm, the female shrimps will be pregnant with eggs and the male shrimps will grow large and long "blue claws" (second walking legs) and other sexual maturity phenomena. Unlike the large-scale mortality incidents that occur during aquaculture, there is no obvious mortality in Macrobrachium rosenbergii after contracting IPS. In general, the food intake decreases, but the phenomenon of "eating a lot but not growing" may also occur, which seriously affects the output and benefits of aquaculture.

[0004] At present, the main pathogenic factors of "iron shrimp syndrome" have not been clearly identified. Epidemiological surveys have found that iron shrimp syndrome will occur after normal shrimp fry and "iron shrimp" are co-cultured, indicating that iron shrimp syndrome is contagious and it is speculated that iron shrimp syndrome may be caused by pathogenic bacteria infection; studies have found that infectious precocity virus (IPV) is related to the occurrence of iron shrimp syndrome, and Macrobrachium rosenbergii infected with IPV will show symptoms of precocious puberty; studies have reported the effects of breeding environment and water quality factors on precocious puberty of Macrobrachium rosenbergii, and the results showed that salinity, breeding density, and temperature significantly affect the gonadal development of Macrobrachium rosenbergii, and also have an inhibitory effect on the growth of Macrobrachium rosenbergii. How to effectively detect and prevent iron shrimp syndrome has become an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide a Macrobrachium rosenbergii circular DNA virus and its detection primers and kit, which are viruses closely related to the slow growth symptoms of Macrobrachium rosenbergii, and provide a research basis for the prevention and treatment of Macrobrachium rosenbergii iron shrimp syndrome. At the same time, the virus detection related technology developed fills the gap of the current lack of nucleic acid molecule detection method that triggers Macrobrachium rosenbergii circular DNA virus, and is suitable for the diagnosis, screening and prevention of Macrobrachium rosenbergii circular DNA virus.

[0006] The technical solution adopted by the present invention to solve its technical problem is: A circular DNA virus of Macrobrachium rosenbergii, classified and named: Macrobrachium rosenbergii circular rep-encoding single-strand DNA virus, deposited by: China General Microbiological Culture Collection Center, deposit number: CGMCC NO:46279, deposit date: December 19, 2024.

[0007] The genome sequence of the virus is shown in SEQ ID No.1.

[0008] The inventors extracted the total DNA of the diseased sample in a conventional manner and used back-to-back primers for PCR amplification to determine the complete sequence of the Macrobrachium rosenbergii circular DNA virus that infects Macrobrachium rosenbergii. The analysis found that its genome is a circular, single-stranded DNA with a total length of 2427nt, containing 2 open reading frames (ORFs), encoding two proteins of 24.2kDa (capsid protein) and 36.6kDa (replication-related protein). According to the results of phylogenetic tree analysis and virus strain classification standards, the Macrobrachium rosenbergii circular DNA virus belongs to a distant strain of the Circoviridae family and is a newly discovered species.

[0009] The application of the Macrobrachium rosenbergii circular DNA virus in the preparation of antibodies or vaccines for preventing and treating Macrobrachium rosenbergii iron shrimp syndrome.

[0010] The vaccine is any one of an inactivated vaccine, a live attenuated vaccine, a subunit vaccine or a genetically engineered vaccine.

[0011] The prevention and control of Macrobrachium rosenbergii iron shrimp syndrome is specifically to prevent and control the slow growth of Macrobrachium rosenbergii iron shrimp syndrome.

[0012] A plasmid contains the genome sequence of the Macrobrachium rosenbergii circular DNA virus.

[0013] A nested PCR detection primer for Macrobrachium rosenbergii circular DNA virus, taking the Macrobrachium rosenbergii circular DNA virus as a detection object, comprising a first-round specific primer for the nested PCR and a second-round specific primer for the nested PCR; The first round of specific primers for nested PCR are: MrCV-F1: 5'-ATGCAGATATGGGGAACTTTTTTTAGAC-3', MrCV-R1: 5'-TTATGCAAGTCCGCTAGAAGCTT-3'; The second round of specific primers for nested PCR are: MrCV-F2: 5'-GCAGCAGATGCGCAGAAGGT-3', MrCV-R2: 5'-ACCGTATTACCCGGCCGAT-3'.

[0014] The invention discloses a nested PCR detection kit for Macrobrachium rosenbergii circular DNA virus. The kit takes the Macrobrachium rosenbergii circular DNA virus as a detection object and comprises a nested PCR amplification reaction system. The nested PCR amplification reaction system (18-25 μL) is composed of: 10-15 mmol / L pH 8.0-8.5 Tris-HCl, 50-80 mmol / L potassium chloride, 5-15 mmol / L magnesium chloride, 1-2 mmol / L dNTP, 0.1-0.2 μmol / L forward primer, 0.1-0.2 μmol / L reverse primer, 1-4 μL DNA template, 1-3 U DNA polymerase, and ddH2O as the balance.

[0015] A fluorescent quantitative PCR detection primer for Macrobrachium rosenbergii circular DNA virus, taking the Macrobrachium rosenbergii circular DNA virus as the detection object, comprising specific primers for fluorescent quantitative PCR; The specific primers for fluorescent quantitative PCR are: MrCV-qF: 5'-GGACCCATGCCTGGAACACCG-3', MrCV-qR: 5'-CGCGCCGGGTAATACGGTCA-3'.

[0016] The invention discloses a fluorescent quantitative PCR detection kit for Macrobrachium rosenbergii circular DNA virus. The kit takes the Macrobrachium rosenbergii circular DNA virus as a detection object and comprises a fluorescent quantitative PCR amplification reaction system. The fluorescent quantitative PCR amplification reaction system (20-50 μL) is composed of: 10-15 mmol / L pH 8.0-8.5 Tris-HCl, 50-80 mmol / L potassium chloride, 5-15 mmol / L magnesium chloride, 5-20 mmol / L dNTP, 0.1-0.2 μmol / L MrCV-qF primer, 0.1-0.2 μmol / L MrCV-qR primer, 0.3-0.5 ul SYBR GREEN fluorescent dye, 1-4 μL DNA template, 1-3 U hot start DNA polymerase, and ddH2O as a balance.

[0017] The kit provided by the invention has strong specificity, high sensitivity and is simple and quick to operate.

[0018] The beneficial effects of the present invention are: (1) The present invention has discovered a Macrobrachium rosenbergii circular DNA virus that is closely related to a slow growth disease. The nucleotide sequence of the virus can be used to prepare primers, probes or kits for detecting the Macrobrachium rosenbergii circular DNA virus, or to prepare antibodies or vaccines against the Macrobrachium rosenbergii circular DNA virus.

[0019] (2) The present invention uses nested PCR and fluorescent quantitative PCR to detect Macrobrachium rosenbergii circular DNA virus. The detection results are specific and easy to judge. It can be used to screen for slow-growing pathogens of Macrobrachium rosenbergii, filling the gap of the current lack of nucleic acid molecule detection methods for Macrobrachium rosenbergii circular DNA virus. The provided primers and kits are suitable for the diagnosis, screening and prevention of Macrobrachium rosenbergii circular DNA virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the phylogenetic tree analysis diagram of the complete nucleotide sequence of MrCV virus; Figure 2 is the Blast comparison result of the partial nucleic acid sequence of MrCV virus in Example 1; Figure 3It is the result diagram of the nested PCR detection of MrCV virus nucleic acid specific sequence in Example 3; a. Typical symptoms of slow growth of Macrobrachium rosenbergii (the shrimp is obviously smaller than normal size or shows symptoms of precocious puberty after 100 days of cultivation); bM: DNA MarkerDL2000; 1-3: Results of the first round of electrophoresis of nested PCR; 1. Homogenate of hepatopancreas of slow-growing Macrobrachium rosenbergii; 2. Homogenate of hepatopancreas of precocious Macrobrachium rosenbergii; 3. Filtrate of normal tissue homogenate of Macrobrachium rosenbergii; 4-6: Results of the second round of electrophoresis of nested PCR; 1. Homogenate of hepatopancreas of slow-growing Macrobrachium rosenbergii; 2. Homogenate of hepatopancreas of precocious Macrobrachium rosenbergii; 6. Homogenate of hepatopancreas of normal Macrobrachium rosenbergii; Figure 4 The specific sequence of MrCV virus nucleic acid detected by fluorescence quantitative PCR in Example 6; a. agarose gel electrophoresis to detect fluorescence quantitative PCR amplification products; (M. DNA Marker DL2000; 1. slow-growing Macrobrachium rosenbergii hepatopancreas homogenate); b. fluorescence quantitative PCR amplification map; c. melting curve of fluorescence quantitative PCR amplification products; Figure 5 : This is the corresponding relationship between the weight of Macrobrachium rosenbergii and the amount of MrCV virus detected by fluorescence quantitative PCR in different farmers in Experimental Example 6; a. The weight of Macrobrachium rosenbergii collected from different farmers; b. The number of virus copies in Macrobrachium rosenbergii collected from different farmers; AE represents different farmers; Figure 6 is the sequence information of SEQ ID No.1; Figure 7 It is the MrCV virus genome structure information; Figure 8 This is the phylogenetic tree result of the amino acid sequence of Rep encoded by MrCV virus; Fig. 9 Coomassie Brilliant Blue and Western blot results of MrCV-Caspid protein expressed in prokaryotes; a. Coomassie Brilliant Blue detection results of pGEX6p1-MrCV Caspid expressed in prokaryotes; M and 1-8 represent Marker, pGEX6p1 prokaryotic expression blank control, pGEX6p1 prokaryotic expression total protein, pGEX6p1 prokaryotic expression product supernatant, pGEX6p1 prokaryotic expression product precipitate, pGEX6p1-MrCV Caspid prokaryotic expression blank control, pGEX6p1-MrCV Caspid prokaryotic expression total protein, pGEX6p1-MrCV Caspid prokaryotic expression product supernatant, pGEX6p1-MrCV Caspid prokaryotic expression product precipitate. b&c. Western blot detection results of pGEX6p1-MrCV Caspid expressed in prokaryotes, b: primary antibody is GST tag antibody, c: primary antibody is MrCV Caspid polyclonal antibody. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below through specific embodiments.

[0022] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified. The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Habor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0023] Embodiment 1: In 2020, Macrobrachium rosenbergii with typical symptoms of iron shrimp syndrome were collected from farmers in Huzhou City, Zhejiang Province about 100 days after cultivation. They were divided into three groups according to their body size and symptoms, namely, those with a body length of less than 5 cm, those with a body length of 5-7 cm and sexual maturity, and those with a body length of more than 7 cm. Some viral nucleic acid sequences were obtained from shrimps with a body length of less than 5 cm and those with precocious puberty. Specific primers were designed for PCR amplification to obtain the full genome sequence of the virus. After sequencing and analysis, it was found that the virus belongs to a single-stranded circular DNA virus encoding replication-related proteins, temporarily named Macrobrachium rosenbergii circular DNA virus (MrCV).

[0024] The analysis found that its genome is circular, single-stranded DNA, with a total length of 2427nt, containing 2 open reading frames (ORFs), encoding two proteins of 24.2kDa (capsid protein) and 36.6kDa (replication-related protein). According to the results of phylogenetic tree analysis and virus strain classification standards, the Macrobrachium rosenbergii circular DNA virus belongs to a distant strain of the Circoviridae family and is a newly discovered species.

[0025] like Figure 1 As shown, the phylogenetic tree analysis results of the full nucleotide sequence of MrCV virus, the red five-pointed star mark represents the Macrobrachium rosenbergii circular DNA virus disclosed in the present invention, and its position shows that it is closely related to the Circoviridae virus and is on an independent branch, belonging to a distant strain of the Circoviridae family.

[0026] Embodiment 2: Acquisition of viral nucleic acid sequences: (1) Hepatopancreas tissues of Macrobrachium rosenbergii with slow growth symptoms (a phenotype of iron shrimp syndrome) were removed and homogenized on ice. The homogenate was diluted with TNMC buffer (50mM Tris-HCl pH 7.5, 100mM NaCl, 10mM CaCl2, 1mM MgCl2) at a ratio of 1:4, centrifuged at 12000rpm for 30min, the supernatant was collected, filtered through a 0.45μm bacterial filter, and the filtrate was treated with RNaseA and DNaseI to remove most of the host's own nucleic acid (background nucleic acid) in the sample. The reaction conditions were 37℃ water bath for 3h, and then 75℃ water bath for 10min to inactivate RNaseA and DNaseI. Viral nucleic acid was extracted using QIAamp DNA Mini Kit (QIAGEN, Germany).

[0027] (2) DNA (20 μL) extracted from the sample was added to the reaction mixture, denatured at 94°C for 3 min, cooled on ice for 2 min, and placed in a 37°C water bath for 1 h after adding 0.5 μL 3'-5'exo-Klenow DNA Polymerase (NEB). The denaturation, annealing and extension process was repeated once to fill in the DNA. The primer used was FR26V-N5'-GCCGGAGCTCTGCAGATATCNNNNNN-3' (SEQ ID No. 2), and the reaction product was used as a template for subsequent amplification.

[0028] Double-stranded DNA filling system Element Dosage (μL) 10×Klenow Buffer 3 dNTP(2.5mM) 4 FR26RV-N Primer 2 DNA template 20 Klenow DNA Polymerase 1 Total reaction system 30 .

[0029] (3) The double-stranded DNA was filled with FR20RV single primer for random PCR amplification, and the product was purified using a PCR product purification kit (Qingke). The sequence of FR20RV single primer is 5'-GCCGGAGCTCTGCAGATATC-3' (SEQ ID No. 3).

[0030] PCR reaction procedure:

[0031] (4) After the PCR amplification product was purified, the primer was removed with restriction endonuclease EcoRV. The target fragments with a size range of 500 to 1500 bp were recovered and ligated to pSIMPLE 19EcoRV / BAP Vector (Takara). The ligated products were transformed into E. coli TOP10 competent cells. After PCR identification, positive clones with fragments of 500 bp or more were selected for sequencing.

[0032] (5) The sequencing results were compared with the nucleotide sequence and the amino acid sequence of the frame reading translation using the Blastn and Blastx tools in the NCBI database. Based on the sequence comparison results, a 651nt segment was obtained that had about 49.41% homology with the partial amino acid sequence of the Niminivirus capsid protein Caspid in GenBank and about 30.22% homology with the partial amino acid sequence of the capsid protein Caspid of the circovirus sp. This sequence was the partial nucleic acid sequence of the virus. The comparison results of the partial nucleic acid sequence of the virus are shown in Figure 2 , indicating that the virus is a single-stranded circular DNA virus.

[0033] Example 3: Nested PCR detection of Macrobrachium rosenbergii circular DNA virus Using bioinformatics methods and the viral nucleic acid sequence information (SEQ ID No. 1) obtained above, specific primers were designed for virus detection.

[0034] The first round of specific primers for nested PCR are: MrCV-F1: 5'-ATGCAGATATGGGGAACTTTTTTTAGAC-3' (SEQ ID No. 4), MrCV-R1: 5'-TTATGCAAGTCCGCTAGAAGCTT-3' (SEQ ID No. 5); the amplified target sequence is 651 bp.

[0035] The second round of specific primers for nested PCR are: MrCV-F2: 5'-GCAGCAGATGCGCAGAAGGT-3' (SEQ ID No. 6), MrCV-R2: 5'-ACCGTATTACCCGGCGCGAT-3' (SEQ ID No. 7); amplified target sequence of 341 bp.

[0036] (1) Preparation of positive samples: Using MrCV-F1 and MrCV-R1 as specific primers and virus-infected tissue DNA as template, a 651 bp target sequence (belonging to Caspid protein) was amplified by ordinary PCR, and a positive standard plasmid molecule containing a 651 bp specific sequence was constructed using gene cloning technology. The standard plasmid used was T vector, provided by Dalian Baosheng Biological Takara Co., Ltd. 1 ng was used as the positive template.

[0037] (2) DNA extraction of the sample to be tested: 20-30 mg of virus-infected Macrobrachium rosenbergii hepatopancreas tissue was homogenized, 200 μL of TE buffer (0.05 mol / L Tris-HCl, 0.02 mol / L EDTA) was added, and 400 μL of guanidine thiocyanate lysis buffer (5 mol / L guanidine thiocyanate, 0.05 mol / L Tris-HCl, 0.02 mol / L EDTA, 1.3% Triton-100), vortex to mix; add 600 μL phenol: chloroform: isoamyl alcohol (25:24:1) to the centrifuge tube, vortex vigorously for 15 seconds, and centrifuge at 13000rpm for 10 minutes; transfer the supernatant to a new 1.5mL centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1), vortex for 15 seconds, and centrifuge at 13000rpm for 10 minutes, transfer the supernatant to a new 1.5mL centrifuge tube, add 0.8 times the volume of isopropanol, and centrifuge at 12000rpm for 10 minutes; discard the isopropanol and wash the precipitate at the bottom of the centrifuge tube once with 1mL of 70% ethanol, place the centrifuge tube at room temperature for 10-15 minutes to dry, then add 100μLTE buffer to dissolve the DNA, and place at -20℃ for use.

[0038] (3) First round of nested PCR: The reaction system was 25 μL, and the reaction solution included: 15 mmol / L pH 8.5 Tris-HCl, 80 mmol / L potassium chloride, 15 mmol / L magnesium chloride, 2 mmol / L dNTP, 0.2 μmol / L MrCV-F1 primer, 0.2 μmol / L MrCV-R1 primer, 2 μL of the DNA template obtained in (2) above, and 2 U DNA polymerase (Takara, Japan).

[0039] (4) Second round of nested PCR: The reaction system was 25 μL, and the reaction solution included: 15 mmol / L pH 8.5 Tris-HCl, 80 mmol / L potassium chloride, 15 mmol / L magnesium chloride, 2 mmol / L dNTP, 0.2 μmol / L MrCV-F2 primer, 0.2 μmol / L MrCV-R2 primer, 2 μL of the PCR product obtained in (3) above, and 2 U DNA polymerase (Takara, Japan).

[0040] (4) Amplification reaction conditions and detection: The reaction conditions for the first round of nested PCR amplification were: 94°C pre-denaturation for 3 min; 94°C for 30 s, 53°C for 30 s, 72°C for 1 min, 35 cycles; and 72°C extension for 10 min.

[0041] The reaction conditions of the second round of nested PCR amplification were as follows: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s; and extension at 72°C for 10 min.

[0042] 10 μL of the amplified product was detected by 1% agarose gel electrophoresis.

[0043] (5) Result judgment: If a single amplified band of 341 bp appears, it means that the sample contains MrCV; otherwise, the sample does not contain MrCV.

[0044] Verification results can be found in Figure 3 , an obvious specific band was amplified in the infected shrimp tissue, and the fragment size was 341bp, which was consistent with expectations, while the corresponding fragment was not amplified in the healthy shrimp tissue samples, indicating that the sequence only came from the shrimp tissue after infection.

[0045] Embodiment 4: The difference between this embodiment and embodiment 3 is that: (3) First round of nested PCR: The reaction system is 18 μL, and the reaction solution includes: 10 mmol / L pH 8.5 Tris-HCl, 50 mmol / L potassium chloride, 5 mmol / L magnesium chloride, 1 mmol / L dNTP, 0.1 μmol / L MrCV-F1 primer, 0.1 μmol / L MrCV-R1 primer, 2 μL of the DNA template obtained in (2) above, and 1 U DNA polymerase; (4) Second round of nested PCR: The reaction system is 18 μL, and the reaction solution includes: 10 mmol / L pH 8.5 Tris-HCl, 50 mmol / L potassium chloride, 5 mmol / L magnesium chloride, 1 mmol / L dNTP, 0.1 μmol / L MrCV-F2 primer, 0.1 μmol / L MrCV-R2 primer, 1 μL of the PCR product obtained in (3) above, and 1 U DNA polymerase.

[0046] Embodiment 5: The difference between this embodiment and embodiment 3 is that: (3) First round of nested PCR: The reaction system is 20 μL, and the reaction solution includes: 15 mmol / L pH 8.0 Tris-HCl, 80 mmol / L potassium chloride, 5 mmol / L magnesium chloride, 2 mmol / L dNTP, 0.2 μmol / L MrCV-F1 primer, 0.2 μmol / L MrCV-R1 primer, 4 μL of the DNA template obtained in (2) above, and 3 U DNA polymerase; (4) Second round of nested PCR: The reaction system is 20 μL, and the reaction solution includes: 15 mmol / L pH 8.0 Tris-HCl, 80 mmol / L potassium chloride, 15 mmol / L magnesium chloride, 2 mmol / L dNTP, 0.2 μmol / L MrCV-F2 primer, 0.2 μmol / L MrCV-R2 primer, 4 μL of the PCR product obtained in (3) above, and 3 U DNA polymerase.

[0047] Example 6: Fluorescence quantitative PCR detection of Macrobrachium rosenbergii circular DNA virus Using bioinformatics methods and the viral nucleic acid sequence information (SEQ ID No. 1) obtained above, specific primers were designed for virus detection.

[0048] The specific primers for fluorescent quantitative PCR are: MrCV-qF: 5'-GGACCCATGCCTGGAACACCG-3' (SEQ ID No. 8), MrCV-qR: 5'-CGCGCCGGGTAATACGGTCA-3' (SEQ ID No. 9). The amplified target sequence is 144 bp.

[0049] (1) Preparation of positive control sample and standard solution: The positive control standard plasmid is the same as the positive sample in Example 3 at 100 ng / ul. When used, the standard plasmid is diluted with RNase-free water to a concentration of 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 The standard solution with 100 copies / μl was used as the reaction template for the preparation of the standard curve of positive samples.

[0050] (2) The method for extracting DNA from the test sample is the same as that in Example 3.

[0051] (3) Negative control sample: DNA from the hepatopancreas tissue of healthy Macrobrachium rosenbergii 500-1000 ng / ul. The same DNA as the sample to be tested will be used in subsequent tests.

[0052] (4) Fluorescence quantitative PCR amplification reaction system: The reaction system is 20 μL, and the reaction solution includes: 10 mmol / L pH 8.5 Tris-HCl, 50 mmol / L potassium chloride, 5 mmol / L magnesium chloride, 5 mmol / L dNTP, 0.2 μmol / L MrCV-qF primer, 0.2 μmol / L MrCV-qR primer, 0.5 ul SYBR GREEN fluorescent dye, 2 μL DNA template, and 2 U hot start DNA polymerase (Takara, Japan).

[0053] (6) Place the added PCR reagent tube into a fluorescent quantitative PCR instrument for amplification. The reaction cycle program is: pre-denaturation at 95°C for 30 seconds; 95°C for 10 seconds, 60°C for 30 seconds, and 40 cycles.

[0054] (7) The fluorescence PCR instrument collects the fluorescence signal, and the computer software automatically generates the CT value of each sample. The standard curve of the positive plasmid template is then prepared to obtain the virus content of the sample.

[0055] Take 10 μL of the above PCR amplification product and detect it by 1% agarose gel electrophoresis to determine whether the sample contains MrCV; the specific determination is: detect the amplification product by gel electrophoresis, observe the electrophoresis results under ultraviolet light, if a single amplification band of 144 bp appears, it means that the sample contains MrCV; otherwise, the sample does not contain MrCV.

[0056] The results of the fluorescence quantitative PCR primers and detection method are shown in Figure 4 Specific nucleic acid fragments were detected in the filtrate of infected shrimp tissue homogenate. The electrophoresis detection and dissolution curve of the amplified products showed that the detection specificity was good.

[0057] Embodiment 7: The difference between this embodiment and embodiment 6 is that: (4) Fluorescence quantitative PCR amplification reaction system: The reaction system is 30 μL, and the reaction solution includes: 10 mmol / L pH 8.5 Tris-HCl, 50 mmol / L potassium chloride, 5 mmol / L magnesium chloride, 5 mmol / L dNTP, 0.1 μmol / L MrCV-qF primer, 0.1 μmol / L MrCV-qR primer, 0.3 μl SYBR GREEN fluorescent dye, 1 μL DNA template, and 1 U hot start DNA polymerase.

[0058] Embodiment 8: The difference between this embodiment and embodiment 6 is that: (4) Fluorescence quantitative PCR amplification reaction system: The reaction system is 50 μL, and the reaction solution includes: 15 mmol / L pH 8.0 Tris-HCl, 80 mmol / L potassium chloride, 15 mmol / L magnesium chloride, 20 mmol / L dNTP, 0.2 μmol / L MrCV-qF primer, 0.2 μmol / L MrCV-qR primer, 0.5 ul SYBR GREEN fluorescent dye, 4 μL DNA template, and 3 U hot start DNA polymerase.

[0059] The content of the virus in Macrobrachium rosenbergii from different farmers was detected by using specific primers for fluorescence quantitative PCR. Macrobrachium rosenbergii from different farmers were collected, the hepatopancreas was taken after weighing, and the total DNA was extracted as a template for subsequent quantitative PCR. The reaction system of fluorescence quantitative PCR was: 12.5μL SYBR Green reaction mixture (TB Green Premix Ex Taq II, Takara, Japan), 1μL each of 10μM MrCV-qF and MrCV-qR primers, 2.5μL of extracted total DNA, and sterile double distilled water was added to 25μL. The reaction conditions were: 95℃30s; 95℃10s, 60℃30s, 40 cycles. The temperature was slowly increased from 60℃ to 95℃ at a rate of 0.5℃ / 5s, and the fluorescence intensity of the sample was continuously measured to obtain the melting curve. The standard curve was established with the positive plasmid control to calculate the content of MrCV in the hepatopancreas of Macrobrachium rosenbergii. The results of using the fluorescence quantitative method to detect the virus in Macrobrachium rosenbergii from different farmers are shown in Figure 5 , showing that the average weight of Macrobrachium rosenbergii from farmer E was the smallest, and the MrCV virus load was the highest.

[0060] Cloning of the whole genome of MrCV and prediction of open reading frames According to the MrCV specific sequence alignment results, the virus is a single-stranded circular DNA virus. According to its genome structure characteristics, back-to-back primers were used to amplify the full-length sequence of the virus. A pair of specific full-length amplification primers were designed based on the sequence information. MrCV-FL-F is 5'-AAGCTTCTAGCGGACTTGCATAA-3' (SEQ ID No. 10), MrCV-FL-R is 5'-AAGCTTGGTCCTTGCCTGATACA-3' (SEQ ID No. 11), and the amplification reaction conditions are: 94℃ pre-denaturation for 3min; 94℃30s, 55℃30s, 72℃2min, 35 cycles. The obtained sequence was connected to the cloning vector through blunt ends, and the MrCV full genome nucleic acid sequence was obtained after sequencing and splicing.

[0061] The complete genome sequence of MrCV is shown in Figure 6The sequencing results showed that the full length of MrCV was 2427 bp, which was consistent with expectations. Based on the full-length sequence of MrCV obtained by sequencing, the open reading frame (ORF) was predicted. The sequence analysis results are shown in Figure 7 The results showed that the virus contained two ORFs, whose predicted functions were capsid protein (Caspid) and replication-related protein (Rep).

[0062] Construction of phylogenetic tree of amino acid sequences of Rep encoded by MrCV virus According to the results of ORF analysis of MrCV sequence, it is predicted that the virus has two ORFs, one of which expresses replication-related protein (Rep). The protein structure is relatively conservative in single-stranded circular DNA viruses and can be used for the construction and analysis of phylogenetic trees. The amino acid sequence of Rep that is closely related to MrCV was retrieved from NCBI, and the phylogenetic tree was constructed by the Neighbour Joining (NJ) method. The analysis results are shown in Figure 8 The results showed that MrCV was on an independent branch and had low similarity with its neighboring virus Niminivirus, and was a new single-stranded circular DNA virus.

[0063] Prokaryotic expression of Caspid protein of MrCV virus and preparation of antibody Capsid protein (Cap) is the main antigenic protein of MrCV, which contains the main antigenic determinant of the virus and plays an important role in the recognition and invasion of the virus. The preparation of anti-Cap antibody is one of the advantageous tools for studying the pathogenic mechanism of MrCV. According to the Caspid nucleic acid sequence of MrCV, the prokaryotic expression recombinant plasmid was constructed. The Caspid sequence of MrCV was amplified by specific primers MrCV-Caspid-F: 5'-CGGGATCCATGCAGATATGGGGAACTTTTTTTAGAC-3' (SEQ ID No.12), MrCV-Caspid-R: 5'-CGGAATTCTTATGCAAGTCCGCTAGAAGCTT-3' (SEQ ID No.13), and the PCR product was subjected to double restriction digestion experiment with pET28a or pGEX6p1 vector, 1 μL each of QuickCut BamH I (Takara) and QuickCut EcoR I (Takara), 3 μL of 10×QuickCut Buffer (Takara), 0.2 μg of PCR product, and 30 μL of ddH2O; the double restriction digestion product was recovered, and the ratio of PCR double restriction digestion product to pET28a or pGEX6p1 vector double restriction digestion product was 5:1, 1 μL of T4 DNA ligase (5 U / μL), 10×T4 DNA ligase buffer (Takara) 2 μL, ddH2O supplemented to 20 μL, ligated the digested products, and transformed the ligated products into E. coli DH5α competent cells; picked clones, verified the correct plasmid by PCR and double enzyme digestion, and then sequenced and verified.

[0064] The correctly sequenced pET28a-MrCV Caspid recombinant plasmid was transformed into expression competent cells, and the antibiotic plates were coated overnight after heat shock; the single clone was picked and cultured overnight in LB liquid medium containing ampicillin (0.1 mg / mL), and the bacterial liquid was added to a new LB liquid medium containing ampicillin (0.1 mg / mL) at a ratio of 1:100. When the OD value was 0.6, 0.5mM IPTG was added, and the cells were cultured overnight at 16°C for large-scale expression. The cell bodies were collected by centrifugation and the cells were broken. The supernatant crude protein was purified to obtain Caspid protein. The obtained Caspid protein was used for the preparation of polyclonal antibodies. After the purified and concentrated Caspid protein was completely emulsified with Freund's adjuvant, New Zealand white rabbits were immunized subcutaneously at multiple points on the back. After three immunizations, blood was collected and serum was separated, and anti-MrCV Caspid antiserum, i.e., polyclonal antibodies, was successfully obtained. The expression culture, purification and immune formation of antibodies for Caspid protein were all entrusted to Shaanxi Bo Minghai Biotechnology Co., Ltd.

[0065] The correctly sequenced pGEX6p1-MrCV Caspid recombinant plasmid was transformed into expression competent cells, and after heat shock, antibiotic plates were coated overnight; a single clone was picked and cultured overnight in a liquid medium containing antibiotics, and the bacterial solution was added to a new LB liquid medium containing ampicillin at a ratio of 1:100. When the OD value reached 0.6, 0.5mM IPTG was added, and the cells were cultured overnight at 16°C for large-scale expression. The bacteria were collected by centrifugation and the cells were broken. The total protein of the uninduced bacterial solution, the total protein of the induced bacterial solution, the supernatant after the broken cells, and the precipitate were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, or western blot detection was performed using GST tag antibody (Affinity Biosciences) (1:5000) and the MrCV Caspid polyclonal antibody prepared above (1:5000) and the corresponding secondary antibody (goat anti-mouse / goat anti-rabbit, Affinity Biosciences). The analysis results are shown in Fig. 9 The results showed that the Caspid protein of MrCV could be correctly expressed and could be detected by GST antibody and Caspid polyclonal antibody at the predicted size position, respectively.

[0066] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A Macrobrachium rosenbergii circular DNA virus, characterized in that: Classification name: Macrobrachium rosenbergii circular rep-encoding single-strand DNA virus, preservation unit: China General Microbiological Culture Collection Administration Center, preservation number: CGMCC NO:46279, preservation date: December 19, 2024.

2. The Macrobrachium rosenbergii circular DNA virus according to claim 1, characterized in that: The genome sequence of the virus is shown in SEQ ID No.

1.

3. Use of the Macrobrachium rosenbergii circular DNA virus as claimed in claim 1 in the preparation of antibodies or vaccines for preventing and treating Macrobrachium rosenbergii iron shrimp syndrome.

4. The use according to claim 3, characterized in that: The vaccine is any one of an inactivated vaccine, a live attenuated vaccine, a subunit vaccine or a genetically engineered vaccine.

5. The use according to claim 3, characterized in that: The prevention and control of Macrobrachium rosenbergii iron shrimp syndrome is specifically to prevent and control the slow growth of Macrobrachium rosenbergii iron shrimp syndrome.

6. A plasmid, characterized in that The invention comprises the genome sequence of the Macrobrachium rosenbergii circular DNA virus according to claim 1.

7. A nested PCR detection primer for Macrobrachium rosenbergii circular DNA virus, characterized in that: The Macrobrachium rosenbergii circular DNA virus of claim 1 is used as the detection object, comprising a first round specific primer of nested PCR and a second round specific primer of nested PCR; The first round of specific primers for nested PCR are: MrCV-F1: 5'-ATGCAGATATGGGGAACTTTTTTTAGAC-3', MrCV-R1: 5'-TTATGCAAGTCCGCTAGAAGCTT-3'; The second round of specific primers for nested PCR are: MrCV-F2: 5'-GCAGCAGATGCGCAGAAGGT-3', MrCV-R2: 5'-ACCGTATTACCCGGCCGAT-3'.

8. A nested PCR detection kit for Macrobrachium rosenbergii circular DNA virus, characterized in that: The Macrobrachium rosenbergii circular DNA virus described in claim 1 is used as the detection object, and includes a nested PCR amplification reaction system, wherein the nested PCR amplification reaction system 18-25 μL is composed of: 10-15 mmol / L pH 8.0-8.5 Tris-HCl, 50-80 mmol / L potassium chloride, 5-15 mmol / L magnesium chloride, 1-2 mmol / L dNTP, 0.1-0.2 μmol / L forward primer of claim 7, 0.1-0.2 μmol / L reverse primer of claim 7, 1-4 μL DNA template, 1-3 U DNA polymerase, and ddH2O as the balance.

9. A fluorescent quantitative PCR detection primer for Macrobrachium rosenbergii circular DNA virus, characterized in that: The Macrobrachium rosenbergii circular DNA virus of claim 1 is used as the detection object, and includes specific primers for fluorescent quantitative PCR; The specific primers for fluorescent quantitative PCR are: MrCV-q: 5'-GGACCCATGCCTGGAACACCG-3', MrCV-qR: 5'-CGCGCCGGGTAATACGGTCA-3'.

10. A fluorescent quantitative PCR detection kit for Macrobrachium rosenbergii circular DNA virus, characterized in that: The Macrobrachium rosenbergii circular DNA virus described in claim 1 is used as the detection object, and includes a fluorescent quantitative PCR amplification reaction system, wherein 20-50 μL of the fluorescent quantitative PCR amplification reaction system is composed of: 10-15 mmol / L pH 8.0-8.5 Tris-HCl, 50-80 mmol / L potassium chloride, 5-15 mmol / L magnesium chloride, 5-20 mmol / L dNTP, 0.1-0.2 μmol / L MrCV-qF primer of claim 9, 0.1-0.2 μmol / L MrCV-qR primer of claim 9, 0.3-0.5 ul SYBR GREEN fluorescent dye, 1-4 μL DNA template, 1-3 U hot start DNA polymerase, and ddH2O as a balance.

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