Macrobrachium rosenbergii circular dna virus and detection primer and kit thereof

Abstract

The application discloses a Macrobrachium rosenbergii circular DNA virus and a detection primer and kit thereof, is classified and named as Macrobrachium rosenbergii circular rep-encoding single-strand DNA virus, is preserved in a preservation unit of China General Microbiological Culture Collection Center, has a preservation number of CGMCC NO:46279, and is preserved on December 19, 2024.The application is closely related to the virus of the slow growth symptom of Macrobrachium rosenbergii, provides a research basis for prevention and treatment of Macrobrachium rosenbergii iron shrimp syndrome, simultaneously develops virus detection related technology, fills the blank that there is no nucleic acid molecule detection method of the Macrobrachium rosenbergii circular DNA virus at present, and is suitable for diagnosis, screening and prevention of the Macrobrachium rosenbergii circular DNA virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, and particularly relates to a Macrobrachium rosenbergii circular DNA virus and a detection primer and kit thereof. BACKGROUND

[0002] Macrobrachium rosenbergii, also known as freshwater long-arm prawn, has become one of the main freshwater aquaculture varieties in China due to its excellent biological characteristics and good breeding benefits. According to the survey, the annual breeding quantity of Macrobrachium rosenbergii in China reached about 28.6 billion in 2022, and the total yield of Macrobrachium rosenbergii reached 196,374 tons in 2023, becoming the largest country in the world in terms of Macrobrachium rosenbergii breeding yield.

[0003] However, high-intensity breeding is accompanied by more challenges. Different growth stages of Macrobrachium rosenbergii will have different diseases, mainly including: larva death syndrome, metamorphosis disorder syndrome, pathogenic enterobacteriosis, ciliates disease, and Vorticella disease in the larva stage; white tail disease in the juvenile stage; and frontal sword white spot disease, gill disease, muscle necrosis disease, slow growth syndrome, white body blister disease, drop star disease, and netting syndrome in the growing stage. Among them, slow growth syndrome has appeared in the Macrobrachium rosenbergii breeding industry since 2010, and its main characteristics are early sexual maturation and slow growth, also known as iron prawn syndrome (IPS). Iron prawn syndrome is a description of the symptoms of early sexual maturation, hard shell, and slow growth in Macrobrachium rosenbergii breeding. Generally, it is characterized by prolonged intermolt interval, significantly slow growth, and sexual maturation of female prawns carrying eggs and male prawns growing large and long "blue claws" (second walking legs) when the prawns grow to 4-6 cm. Unlike large-scale death events in aquaculture, Macrobrachium rosenbergii does not show obvious death after suffering from IPS. Generally, the feeding amount decreases, but there is also the phenomenon of "eating a lot but not growing much", which seriously affects the yield and benefit of breeding.

[0004] At present, the main pathogenic factors causing iron prawn syndrome have not been clearly identified. Epidemiological investigations have found that normal prawn fry and "iron prawn" mixed breeding will cause iron prawn syndrome, indicating that iron prawn syndrome is contagious, and it is speculated that iron prawn syndrome may be caused by pathogenic bacteria infection. Current studies have found that infectious precocity virus (IPV) is related to the occurrence of iron prawn syndrome, and Macrobrachium rosenbergii infected with IPV will show symptoms of early sexual maturation. Studies have reported the effects of breeding environment and water quality factors on the sexual maturation of Macrobrachium rosenbergii, and the results show 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 discover and prevent iron prawn syndrome has become a problem to be solved. SUMMARY

[0005] The present application aims to provide a Macrobrachium rosenbergii circular DNA virus and a detection primer and kit thereof, which is closely related to the growth retardation symptom of Macrobrachium rosenbergii, provides a research basis for the prevention and treatment of Macrobrachium rosenbergii iron shrimp syndrome, and simultaneously develops a virus detection related technology to fill the blank of the nucleic acid molecule detection method of the Macrobrachium rosenbergii circular DNA virus, which is suitable for the diagnosis, screening and prevention of the Macrobrachium rosenbergii circular DNA virus.

[0006] The technical scheme adopted by the present application to solve its technical problems is:

[0007] A Macrobrachium rosenbergii circular DNA virus, classified and named as: Macrobrachium rosenbergii circular rep-encoding single-strand DNA virus, preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC NO:46279 and a preservation date of December 19, 2024.

[0008] The genome sequence of the virus is shown in SEQ ID No. 1.

[0009] The present application discloses a Macrobrachium rosenbergii circular DNA virus, classified and named as: Macrobrachium rosenbergii circular rep-encoding single-strand DNA virus, preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC NO:46279 and a preservation date of December 19, 2024.

[0010] The Macrobrachium rosenbergii circular DNA virus is used for preparing an antibody or a vaccine for preventing and treating Macrobrachium rosenbergii iron shrimp syndrome.

[0011] The vaccine is any one of an inactivated vaccine, a live attenuated vaccine, a subunit vaccine or a genetic engineering vaccine.

[0012] The Macrobrachium rosenbergii iron shrimp syndrome is specifically growth retardation in the Macrobrachium rosenbergii iron shrimp syndrome.

[0013] A plasmid comprising the genome sequence of the Macrobrachium rosenbergii circular DNA virus.

[0014] A nested PCR detection primer of Macrobrachium rosenbergii circular DNA virus, taking the Macrobrachium rosenbergii circular DNA virus as a detection object, comprising a first round of specific primers of nested PCR and a second round of specific primers of nested PCR;

[0015] The first round of specific primers of nested PCR are:

[0016] MrCV-F1: 5'-ATGCAGATATGGGGAACTTTTTTTAGAC-3',

[0017] MrCV-R1: 5'-TTATGCAAGTCCGCTAGAAGCTT-3';

[0018] The second round of specific primers of nested PCR are:

[0019] MrCV-F2: 5'-GCAGCAGATGCGCAGAAGGT-3',

[0020] MrCV-R2: 5'-ACCGTATTACCCGGCGCGAT-3'.

[0021] A nested PCR detection kit of Macrobrachium rosenbergii circular DNA virus, taking the Macrobrachium rosenbergii circular DNA virus as a detection object, comprising a nested PCR amplification reaction system, and the nested PCR amplification reaction system 18-25 μL is composed of: 10-15 mmol / L of Tris-HCl with pH 8.0-8.5, 50-80 mmol / L of potassium chloride, 5-15 mmol / L of magnesium chloride, 1-2 mmol / L of dNTP, 0.1-0.2 μmol / L of forward primer, 0.1-0.2 μmol / L of reverse primer, 1-4 μL of DNA template, 1-3 U of DNA polymerase, and the rest of ddH2O.

[0022] A fluorescent quantitative PCR detection primer of Macrobrachium rosenbergii circular DNA virus, taking the Macrobrachium rosenbergii circular DNA virus as a detection object, comprising specific primers of fluorescent quantitative PCR;

[0023] The specific primers of fluorescent quantitative PCR are:

[0024] MrCV-qF: 5'-GGACCCATGCCTGGAACACCG-3',

[0025] MrCV-qR: 5'-CGCGCCGGGTAATACGGTCA-3'.

[0026] A fluorescent quantitative PCR detection kit for Macrobrachium rosenbergii circular DNA virus, which 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 muL is composed of: 10-15 mmol / L of 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 mu mol / L MrCV-qF primer, 0.1-0.2 mu mol / L MrCV-qR primer, 0.3-0.5 ul SYBR GREEN fluorescent dye, 1-4 muL DNA template, 1-3 U hot start DNA polymerase, and ddH2O in a residual amount.

[0027] The kit provided by the application has high specificity and sensitivity, and is simple, fast and convenient to operate.

[0028] The application has the following beneficial effects:

[0029] (1) The application finds a Macrobrachium rosenbergii circular DNA virus closely related to the slow growth syndrome of Macrobrachium rosenbergii, and 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.

[0030] (2) The application applies nested PCR and fluorescent quantitative PCR to detect the Macrobrachium rosenbergii circular DNA virus, and the detection result is specific and easy to judge, and can be used for screening the pathogen of the slow growth of Macrobrachium rosenbergii, fills the blank of the nucleic acid molecular detection method of the Macrobrachium rosenbergii circular DNA virus at present, and the primers and kits provided are suitable for the diagnosis, screening and prevention of the Macrobrachium rosenbergii circular DNA virus. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a phylogenetic tree analysis diagram of the whole nucleotide sequence of the MrCV virus;

[0032] Figure 2 is a Blast comparison result of part of the nucleic acid sequence of the MrCV virus in Example 1;

[0033] Figure 3is a result chart of nested PCR detection of MrCV virus nucleic acid specific sequence in Example 3; a. Typical symptoms of slow growth of Macrobrachium rosenbergii (shrimp is significantly smaller than normal size or appears precocious symptoms when cultured for 100 days); b. M: DNA Marker DL2000; 1-3: first round electrophoresis result of nested PCR; 1. Homogenate of hepatopancreas of slow growth Macrobrachium rosenbergii; 2. Homogenate of hepatopancreas of precocious Macrobrachium rosenbergii; 3. Homogenate filtrate of normal Macrobrachium rosenbergii tissue; 4-6: second round electrophoresis result of nested PCR; 1. Homogenate of hepatopancreas of slow growth Macrobrachium rosenbergii; 2. Homogenate of hepatopancreas of precocious Macrobrachium rosenbergii; 6. Homogenate of hepatopancreas of normal Macrobrachium rosenbergii;

[0034] Figure 4 is a result chart of fluorescence quantitative PCR detection of MrCV virus nucleic acid specific sequence in Example 6; a. Agarose gel electrophoresis detection of fluorescence quantitative PCR amplification product; (M. DNA Marker DL2000; 1. Homogenate of hepatopancreas of slow growth Macrobrachium rosenbergii); b. Fluorescence quantitative PCR amplification chart; c. Dissolution curve of fluorescence quantitative PCR amplification product;

[0035] Figure 5 is a chart of corresponding relationship between body weight of Macrobrachium rosenbergii and MrCV virus amount in different farmers in Example 6; a. Body weight of Macrobrachium rosenbergii collected in different farmers; b. Virus copy number in Macrobrachium rosenbergii collected in different farmers; A-E represent different farmers;

[0036] Figure 6 is sequence information of SEQ ID No. 1;

[0037] Figure 7 is genomic structure information of MrCV virus;

[0038] Figure 8 is a result chart of system evolution tree of Rep amino acid sequence coded by MrCV virus;

[0039] Figure 9The results of Coomassie brilliant blue and Western blot detection of prokaryotic expression of MrCV-Caspid protein; a. The results of Coomassie brilliant blue detection of prokaryotic expression of pGEX6p1-MrCV Caspid; M and 1-8 represent Marker, pGEX6p1 prokaryotic expression blank control, pGEX6p1 prokaryotic expression total protein, pGEX6p1 prokaryotic expression supernatant, pGEX6p1 prokaryotic expression precipitate, pGEX6p1-MrCV Caspid prokaryotic expression blank control, pGEX6p1-MrCV Caspid prokaryotic expression total protein, pGEX6p1-MrCV Caspid prokaryotic expression supernatant, and pGEX6p1-MrCV Caspid prokaryotic expression precipitate. b and c. The results of Western blot detection of prokaryotic expression of pGEX6p1-MrCV Caspid, b: the primary antibody is GST tag antibody, and c: the primary antibody is MrCV Caspid polyclonal antibody. DETAILED DESCRIPTION

[0040] The technical solutions of the present application are further described below through specific examples.

[0041] In the present application, unless specified, the raw materials and equipment used can be purchased from the market or commonly used in the art. The methods in the following examples are conventional methods in the art, unless otherwise specified. The experimental methods not specified in the examples are generally carried out under conventional conditions, such as the conditions 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.

[0042] Example 1

[0043] In 2020, Macrobrachium rosenbergii with typical symptoms of iron shrimp syndrome when cultured for about 100 days were collected from a breeder in Huzhou, Zhejiang Province, and divided into three groups according to body size and symptoms, i.e., body length less than 5 cm, body length 5-7 cm and sexual maturity, and body length greater than 7 cm. Partial viral nucleic acid sequences were obtained from the body length less than 5 cm and sexually precocious sick shrimp, and specific primers were designed to amplify the whole genome sequence of the virus by PCR for sequencing analysis. It was found that the virus belongs to a single-stranded circular DNA virus encoding a replication-associated protein, which is temporarily named as Macrobrachium rosenbergii circular DNA virus (MrCV).

[0044] The analysis found that its genome is circular, single-stranded DNA, full-length 2427 nt, containing 2 open reading frames (ORF), encoding size 24.2 kDa (capsid protein) and 36.6 kDa (replication associated protein) two proteins. According to the phylogenetic tree analysis results and virus strain classification standard, the Macrobrachium rosenbergii circular DNA virus belongs to a distant strain of the Circoviridae family, and is a newly discovered species.

[0045] As shown in Figure 1 the phylogenetic tree analysis results of the full nucleotide sequence of the MrCV virus, the red pentagram mark represents the Macrobrachium rosenbergii circular DNA virus disclosed in the present application, and the position shows that it is relatively close to the Circoviridae virus and is on an independent branch, belonging to a distant strain of the Circoviridae family.

[0046] Example 2:

[0047] Obtaining of viral nucleic acid sequence:

[0048] (1) The Macrobrachium rosenbergii hepatopancreas tissue with slow growth symptoms (a phenotype of iron shrimp syndrome) was taken out and homogenized on ice. The homogenate was diluted with TNMC buffer ((50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM CaCl2, 1 mM MgCl2)) at a ratio of 1:4, centrifuged at 12000 rpm for 30 min, and the supernatant was taken, filtered through a 0.45 μm bacterial filter, and treated with RNase A and DNase I to remove most of the host's own nucleic acid (background nucleic acid) in the sample. The reaction conditions were 37°C water bath for 3 h, and then 75°C water bath for 10 min to inactivate RNase A and DNase I. The viral nucleic acid was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany) kit.

[0049] (2) The DNA (20 μL) extracted from the sample was added to the reaction system mixture, denatured at 94°C for 3 min, cooled on ice for 2 min, and then 0.5 μL 3'-5'exo-Klenow DNA Polymerase (NEB) was added and placed in a 37°C water bath for 1 h. The denaturation, annealing and extension processes were repeated once to fill in the DNA. The primer used was FR26V-N 5'-GCCGGAGCTCTGCAGATATCNNNNNN-3' (SEQ ID No. 2), and the reaction product was used as the subsequent amplification template.

[0050] Double-stranded DNA filling system

[0051] Ingredients Amount (μL) 10 x Klenow Buffer 3 dNTP (2.5 mM) 4 FR26RV-N primer 2 DNA template 20 Klenow DNA Polymerase 1 Total reaction 30 .

[0052] (3) The patched double-stranded DNA was subjected to random PCR amplification using the FR20RV single primer, and the product was purified using a PCR product purification kit (Qiagen). The sequence of the FR20RV single primer was 5'-GCCGGAGCTCTGCAGATATC-3' (SEQ ID No. 3).

[0053] PCR reaction program:

[0054]

[0055]

[0056] (4) After the PCR amplification product was purified, the primer was removed using the restriction endonuclease EcoR V. The target fragment with a size ranging from 500 to 1500 bp was recovered and ligated to pSIMPLE 19 EcoRV / BAP Vector (Takara), and the ligation product was transformed into E. coli TOP10 competent cells. After PCR identification, positive clones with different fragment sizes of more than 500 bp were selected for sequencing.

[0057] (5) The sequencing results were subjected to nucleotide sequence and frame reading translated amino acid sequence alignment in the NCBI database using the Blastn and Blastx tools. Based on the sequence alignment results, a 651 nt sequence was obtained, which had about 49.41% homology with the Caspid part of the amino acid sequence of the Niminivirus coat protein in GenBank and about 30.22% homology with the Caspid part of the amino acid sequence of the Circovirus sp. coat protein, and this sequence was the partial nucleic acid sequence of the virus. The alignment results of the partial nucleic acid sequence of the virus are shown in Figure 2 , indicating that the virus belongs to a single-stranded circular DNA virus.

[0058] Example 3: Nested PCR detection of Macrobrachium rosenbergii circular DNA virus

[0059] Bioinformatics methods and the above-mentioned viral nucleic acid sequence information (SEQ ID No. 1) were used to design specific primers for virus detection.

[0060] The first round of specific primers for nested PCR were:

[0061] MrCV-F1: 5'-ATGCAGATATGGGGAACTTTTTTTAGAC-3' (SEQ ID No. 4), and MrCV-R1: 5'-TTATGCAAGTCCGCTAGAAGCTT-3' (SEQ ID No. 5); the amplified target sequence was 651 bp.

[0062] The second round of specific primers for nested PCR are:

[0063] MrCV-F2: 5'-GCAGCAGATGCGCAGAAGGT-3' (SEQ ID No. 6),

[0064] MrCV-R2: 5'-ACCGTATTACCCGGCGCGAT-3' (SEQ ID No. 7); amplified target sequence 341bp.

[0065] (1) Preparation of positive samples: Using MrCV-F1 and MrCV-R1 as specific primers and viral-infected tissue DNA as template, a target sequence (belonging to the Caspid protein) was amplified by conventional PCR. A positive standard plasmid molecule containing the 651bp specific sequence was constructed using gene cloning technology. The standard plasmid used was a T vector provided by Takara Bio Inc. of Dalian Baosheng Biotechnology Co., Ltd. 1 ng was used as the positive template.

[0066] (2) Extraction of DNA from the sample to be tested: Take 20-30 mg of hepatopancreatic tissue from virus-infected giant freshwater prawns, homogenize it, add 200 μL of TE buffer (0.05 mol / L Tris-HCl, 0.02 mol / L EDTA), homogenize again, and add 400 μL of guanidine isothiocyanate lysis buffer (5 mol / L guanidine isothiocyanate, 0.05 mol / L Tris-HCl, 0.02 mol / L EDTA, 1.3%). Triton-100 solution was used to mix the DNA by vortexing. Then, 600 μL of phenol:chloroform:isoamyl alcohol (25:24:1) was added to a centrifuge tube, vortexed vigorously for 15 seconds, and centrifuged at 13000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of chloroform:isoamyl alcohol (24:1) was added. The mixture was vortexed for 15 seconds and centrifuged at 13000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 mL centrifuge tube, and 0.8 times the volume of isopropanol was added. The mixture was centrifuged at 12000 rpm for 10 minutes. After discarding the isopropanol, the precipitate at the bottom of the centrifuge tube was washed once with 1 mL of 70% ethanol. The centrifuge tube was then left to dry at room temperature for 10–15 minutes. Finally, 100 μL of TEL buffer was added to dissolve the DNA, and the tube was stored at -20°C for later use.

[0067] (3) First round nested PCR: The reaction system was 25 μL, and the reaction solution was prepared to include 15 mmol / L of 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 the above (2), and 2 U of DNA polymerase (Takara, Japan).

[0068] (4) Second round nested PCR: The reaction system was 25 μL, and the reaction solution was prepared to include 15 mmol / L of 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 the above (3), and 2 U of DNA polymerase (Takara, Japan).

[0069] (4) Amplification reaction conditions and detection: The reaction conditions of the first round nested PCR amplification were as follows: 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.

[0070] The reaction conditions of the second round nested PCR amplification were as follows: 94°C pre-denaturation for 3 min; 94°C for 30 s, 57°C for 30 s, 72°C for 30 s, 35 cycles; and 72°C extension for 10 min.

[0071] 10 μL of the amplification product was detected by 1% agarose electrophoresis.

[0072] (5) Result judgment: If a single amplification band of 341 bp appeared, it indicated that the sample contained MrCV; otherwise, the sample did not contain MrCV.

[0073] The verification results are shown in Table 1. Figure 3 An obvious specific band was amplified in the infected shrimp tissue, the fragment size was 341 bp, which was consistent with the expectation, and no corresponding fragment was amplified in the healthy shrimp tissue sample, indicating that the sequence only came from the infected shrimp tissue.

[0074] Example 4:

[0075] The difference between this example and Example 3 is as follows:

[0076] (3) The first nested PCR: the reaction system is 18 μL, and the reaction solution is prepared to include 10 mmol / L of 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 the above (2), and 1 U of DNA polymerase;

[0077] (4) The second nested PCR: the reaction system is 18 μL, and the reaction solution is prepared to include 10 mmol / L of 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 the above (3), and 1 U of DNA polymerase.

[0078] Example 5:

[0079] The difference between this example and Example 3 is that:

[0080] (3) The first nested PCR: the reaction system is 20 μL, and the reaction solution is prepared to include 15 mmol / L of 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 the above (2), and 3 U of DNA polymerase;

[0081] (4) The second nested PCR: the reaction system is 20 μL, and the reaction solution is prepared to include 15 mmol / L of 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 the above (3), and 3 U of DNA polymerase.

[0082] Example 6: Fluorescent quantitative PCR detection of Macrobrachium rosenbergii circular DNA virus

[0083] The specific primers for the fluorescent quantitative PCR are designed by using bioinformatics methods and the viral nucleic acid sequence information (SEQ ID No. 1) obtained above.

[0084] The specific primers for the fluorescent quantitative PCR are designed by using bioinformatics methods and the viral nucleic acid sequence information (SEQ ID No. 1) obtained above.

[0085] MrCV-qF: 5'-GGACCCATGCCTGGAACACCG-3' (SEQ ID No. 8),

[0086] MrCV-qR: 5'-CGCGCCGGGTAATACGGTCA-3' (SEQ ID No. 9). The amplified target sequence is 144 bp.

[0087] (1) Preparation of positive control sample and standard solution: the positive control standard plasmid is 100 ng / ul, same as the positive sample in Example 3. When used, the standard plasmid is diluted with ribonuclease-free water, i.e. diluted to a concentration of 1.0 x 10 8 , 1.0 x 10 7 , 1.0 x 10 6 , 1.0 x 10 5 , 1.0 x 10 4 copies / ul, as the reaction template for making the standard curve of the positive sample.

[0088] (2) The DNA extraction method of the sample to be tested is the same as that in Example 3.

[0089] (3) Negative control sample: healthy Macrobrachium rosenbergii liver and pancreas tissue DNA 500-1000 ng / ul. The subsequent use is the same as the DNA of the sample to be tested.

[0090] (4) Fluorescent quantitative PCR amplification reaction system: the reaction system is 20 μL, and the preparation of the reaction solution includes: 10 mmol / L of Tris-HCl with pH 8.5, 50 mmol / L of potassium chloride, 5 mmol / L of magnesium chloride, 5 mmol / L of dNTP, 0.2 μmol / L of MrCV-qF primer, 0.2 μmol / L of MrCV-qR primer, 0.5 ul of SYBR GREEN fluorescent dye, 2 μL of DNA template, 2 U of hot start DNA polymerase (Takara, Japan).

[0091] (6) After adding the sample, the PCR reagent tube is placed in the fluorescent quantitative PCR instrument for amplification, and the reaction cycle program is: 95℃ pre-denaturation for 30s; 95℃ for 10s, 60℃ for 30s, 40 cycles.

[0092] (7) The fluorescent PCR instrument collects the fluorescence signal, and the CT value of each sample is automatically generated by computer software, and the standard curve of the positive plasmid template can be obtained to obtain the virus content of the sample.

[0093] Take 10 μL of the PCR amplification product, 1% agarose gel electrophoresis to detect whether the sample contains MrCV; the judgment is as follows: gel electrophoresis detection of the amplification product, observe the electrophoresis result under the ultraviolet lamp, if a single amplification band of 144 bp appears, it means that the sample contains MrCV; otherwise, the sample does not contain MrCV.

[0094] The results of the verification of the fluorescence quantitative PCR primer and detection method are shown in Table 1. Figure 4 Specific nucleic acid fragments were detected in the filtrate of the infected shrimp tissue homogenate, and the electrophoresis detection and melting curve of the amplification product showed that the specificity of the detection was good.

[0095] Example 7:

[0096] The difference between this example and Example 6 is that:

[0097] (4) The fluorescence quantitative PCR amplification reaction system: the reaction system is 30 μL, and the reaction solution includes: 10 mmol / L of 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 of hot start DNA polymerase.

[0098] Example 8:

[0099] The difference between this example and Example 6 is that:

[0100] (4) The fluorescence quantitative PCR amplification reaction system: the reaction system is 50 μL, and the reaction solution includes: 15 mmol / L of 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 of hot start DNA polymerase.

[0101] Detection of virus content in different farmers' Macrobrachium rosenbergii by fluorescent quantitative PCR Specific primers were used to detect the virus content in different farmers' Macrobrachium rosenbergii. The liver and pancreas of Macrobrachium rosenbergii were weighed and total DNA was extracted as a template for subsequent quantitative PCR. The reaction system of fluorescent quantitative PCR was as follows: 12.5 μL of SYBR Green reaction mixture (TB Green Premix Ex Taq II, Takara, Japan), 1 μL of 10 μM MrCV-qF and MrCV-qR primers, 2.5 μL of extracted total DNA, and sterile double distilled water to make up to 25 μL. The reaction conditions were as follows: 95 °C for 30 s; 95 °C for 10 s, 60 °C for 30 s, 40 cycles. The fluorescence intensity of the sample was continuously measured at a rate of 0.5 °C / 5 s from 60 °C to 95 °C to obtain the melting curve. The standard curve was established by using the positive plasmid control, and the content of MrCV in the liver and pancreas of Macrobrachium rosenbergii was calculated. The results of detecting the virus content in different farmers' Macrobrachium rosenbergii by fluorescent quantitative method are shown in Figure 5 , which shows that the average weight of Macrobrachium rosenbergii in farmer E is the smallest, and the MrCV virus content is the highest.

[0102] Cloning of MrCV full genome and prediction analysis of open reading frame

[0103] According to the alignment results of MrCV specific sequences, the virus is a single-stranded circular DNA virus. According to the structural characteristics of its genome, a pair of specific full-length amplification primers was designed based on the sequence information. MrCV-FL-F is 5'-AAGCTTCTAGCGGACTTGCATAA-3'(SEQ ID No. 10), and MrCV-FL-R is 5'-AAGCTTGGTCCTTGCCTGATACA-3'(SEQ ID No. 11). The amplification reaction conditions are as follows: 94 °C pre-denaturation for 3 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, 35 cycles. The obtained sequence was ligated to the cloning vector after blunt-end ligation, and the MrCV full genome nucleic acid sequence was obtained after sequencing and splicing.

[0104] The MrCV full genome nucleic acid sequence is shown in Figure 6 , and the sequencing results show that the full length of MrCV is 2427 bp, which is consistent with the expectation. According to the full length sequence of MrCV obtained by sequencing, the prediction of open reading frame (ORF) was carried out, and the sequence analysis results are shown in Figure 7 , which shows that the virus contains two ORFs, and the predicted functions are capsid protein (Caspid) and replication associated protein (Rep), respectively.

[0105] Construction of a phylogenetic tree of MrCV virus encoding Rep amino acid sequence

[0106] According to the ORF analysis of MrCV sequence, it is predicted that the virus has two ORFs, one of which expresses replication-associated protein (Rep), which is relatively conserved in single-stranded circular DNA viruses and can be used to construct and analyze phylogenetic trees. By searching the Rep amino acid sequences of sequences closely related to MrCV in NCBI, and constructing a phylogenetic tree by Neighbour Joining (NJ), the analysis results are shown in Figure 8 , which shows that MrCV is on an independent branch, has low similarity with its adjacent virus Niminivirus, and is a new single-stranded circular DNA virus.

[0107] Prokaryotic expression of Caspid protein of MrCV virus and preparation of antibody

[0108] 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. Preparation of anti-Cap antibody is one of the favorable tools for studying the pathogenic mechanism of MrCV. According to the Caspid nucleic acid sequence of MrCV, a 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 double-digested with pET28a or pGEX6p1 vector. QuickCut BamH I (Takara) and QuickCut EcoR I (Takara) were each 1 μL, 10×QuickCut Buffer (Takara) was 3 μL, PCR product was 0.2 μg, and ddH2O was supplemented to 30 μL. The double-digested product was recovered, and the ratio of PCR double-digested product to pET28a or pGEX6p1 vector double-digested product was 5:1, T4 DNA ligase (5 U / μL) was 1 μL, 10×T4 DNA ligase Buffer (Takara) was 2 μL, and ddH2O was supplemented to 20 μL. The ligase-digested product was transformed into E. coli DH5α competent cells. The clones were picked, and the correct plasmid was verified by PCR and double digestion and sequencing.

[0109] The correctly sequenced pET28a-MrCV Caspid recombinant plasmid was transformed into expression competent cells, and after heat shock, the cells were plated on an antibiotic plate overnight; a single colony was picked and cultured in LB liquid medium containing ampicillin (0.1 mg / mL) overnight, and then the bacterial solution was added to new LB liquid medium containing ampicillin (0.1 mg / mL) at a ratio of 1:100, and when the OD value reached 0.6, 0.5 mM IPTG was added, and the cells were cultured at 16°C overnight for mass expression. The cells were collected by centrifugation and the cells were broken, and the supernatant was obtained after purification to obtain the Caspid protein, and the obtained Caspid protein was used for the preparation of polyclonal antibody, and after complete emulsification of the purified and concentrated Caspid protein with Freund's adjuvant, the New Zealand white rabbits were immunized subcutaneously on the back, and after three immunizations, blood was collected and serum was separated, and the anti-MrCV Caspid antiserum, i.e., polyclonal antibody, was successfully obtained. The Caspid protein expression culture, purification and antibody formation were all entrusted to Shaanxi Bominghai Biotechnology Co., Ltd.

[0110] The correctly sequenced pET28a-MrCV Caspid recombinant plasmid was transformed into expression competent cells, and after heat shock, the cells were plated on an antibiotic plate overnight; a single colony was picked and cultured in LB liquid medium containing ampicillin (0.1 mg / mL) overnight, and then the bacterial solution was added to new LB liquid medium containing ampicillin (0.1 mg / mL) at a ratio of 1:100, and when the OD value reached 0.6, 0.5 mM IPTG was added, and the cells were cultured at 16°C overnight for mass expression. The cells were collected by centrifugation and the cells were broken, and the supernatant was obtained after purification to obtain the Caspid protein, and the obtained Caspid protein was used for the preparation of polyclonal antibody, and after complete emulsification of the purified and concentrated Caspid protein with Freund's adjuvant, the New Zealand white rabbits were immunized subcutaneously on the back, and after three immunizations, blood was collected and serum was separated, and the anti-MrCV Caspid antiserum, i.e., polyclonal antibody, was successfully obtained. The Caspid protein expression culture, purification and antibody formation were all entrusted to Shaanxi Bominghai Biotechnology Co., Ltd. Figure 9 , and 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, respectively.

[0111] The above-described embodiments are only a preferred scheme of the present application, and do not limit the present application in any form, and other variants and modifications can be made without exceeding the technical scheme recited in the claims.

Claims

1. A circular DNA virus of the giant freshwater prawn, characterized in that, Classified and named: Giant freshwater prawn circular DNA virus Macrobrachium rosenbergiicircular rep-encoding single-strand DNA virus Depository institution: China General Microbiological Culture Collection Center, accession number: CGMCC NO:46279, deposit date: December 19, 2024.

2. A plasmid, characterized in that, The genome sequence of the giant freshwater shrimp circular DNA virus as described in claim 1 is included.

3. A nested PCR detection primer for a circular DNA virus in the giant freshwater prawn, characterized in that, Using the circular DNA virus of giant freshwater prawn as described in claim 1 as the detection target, the method includes a first round of specific primers for nested PCR and a second round of specific primers for 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'.

4. A nested PCR detection kit for a circular DNA virus in giant freshwater prawns, characterized in that, Using the circular DNA virus of Macrobrachium rosenbergii as described in claim 1 as the detection target, the method includes a nested PCR amplification reaction system. The nested PCR amplification reaction system (18-25 μL) consists 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 dNTPs, 0.1-0.2 μmol / L forward primer of claim 3, 0.1-0.2 μmol / L reverse primer of claim 3, 1-4 μL DNA template, 1-3 U DNA polymerase, and the remainder ddH2O.

5. A primer for the real-time quantitative PCR detection of a circular DNA virus in the giant freshwater prawn, characterized in that, Using the circular DNA virus of giant freshwater prawn as described in claim 1 as the detection target, including specific primers for real-time PCR; The specific primers for quantitative real-time PCR are: MrCV-qF: 5'-GGACCCATGCCTGGAACACCG-3', MrCV-qR: 5'-CGCGCCGGGTAATACGGTCA-3'.

6. A real-time PCR detection kit for a circular DNA virus in giant freshwater prawns, characterized in that, Using the circular DNA virus of *Macrobrachium rosenbergii* as described in claim 1 as the detection target, the method includes a quantitative real-time PCR amplification reaction system. The quantitative real-time PCR amplification reaction system (20-50 μL) comprises: 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 dNTPs, 0.1-0.2 μmol / L MrCV-qF primers of claim 5, 0.1-0.2 μmol / L MrCV-qR primers of claim 5, 0.3-0.5 μL SYBR Green fluorescent dye, 1-4 μL DNA template, 1-3 U hot-start DNA polymerase, and the remainder ddH2O.

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