A nucleotide sequence and its application

Abstract

The present invention provides a nucleotide sequence and its application. The nucleotide sequence is shown as SEQ ID NO.1. Through this sequence, the gene structure and function related to the proliferation and important regulatory functions of Macrobrachium rosenbergii dicistrovirus type III can be understood, the molecular physiological mechanism and the key points of how it infects the host can be found, and the virus pathogenic or even lethal genes can be searched for. In addition, this nucleotide sequence is crucial for the design of products such as primers, probes and kits for the detection of Macrobrachium rosenbergii dicistrovirus type III, and also has important guiding significance for the subsequent development of related drug products.

Description

Technical Field

[0001] The present invention relates to the technical field of biological genetic engineering, and specifically relates to a nucleotide sequence and its application. Background Art

[0002] The giant freshwater prawn (Macrobrachium rosenbergii), also known as the freshwater long-armed prawn, Thai prawn, Malaysian prawn, etc., is an important freshwater economic cultured shrimp species in China and is also a key seedling-breeding variety for "winning the battle of the seed industry" in Zhejiang Province. In 2021, the national aquaculture area of the giant freshwater prawn was nearly 600,000 mu, and Zhejiang Province provided 60% of the total national giant freshwater prawn fry. In the national-level giant freshwater prawn breeding farms in Zhejiang Province, there are 13 generations of breeding families preserved, ensuring the stable supply of fry and breaking the monopoly of international seed industry giants. However, since 2018, an infectious disease manifested as "muscle white body atrophy" has appeared in the adult giant freshwater prawn aquaculture process, known as "white body disease". This disease can cause a significant decrease in aquaculture production and the phenomenon that the finished shrimp is prone to death during transportation, resulting in large losses for farmers and becoming a relatively serious infectious disease affecting the adult giant freshwater prawn aquaculture today. We discovered a novel Macrobrachium rosenbergii dicistrovirus 3 (MrDV-3) related to the disease from the diseased shrimp. There is no relevant report on this virus at home and abroad, and its viral genome sequence has not been publicly released or applied. According to the comparison with the gene database in NCBI, only two virus sequences with similarities of 81.98% and 83.31% obtained from water samples from the Havel River in Germany and the Yangtze River in China were compared. This indicates that the virus is a completely new virus. Since there have been reports of two other dicistroviruses in Macrobrachium rosenbergii, one is the dicistrovirus-1 from the larvae in the seedling stage of Chinese Macrobrachium rosenbergii, and the other is the dicistrovirus-1 from the liver of Malaysian cultured Macrobrachium rosenbergii. Therefore, in order to prevent confusion among the above three viruses, the dicistrovirus involved in the present invention is specifically referred to as Macrobrachium rosenbergii Dicistrovirus III (MrDV-3). Since this virus is a completely new virus, conducting a detailed study on it is of great significance for subsequent vaccine development, prevention and control of infectious diseases of Macrobrachium rosenbergii, etc. Summary of the Invention

[0003] The purpose of the present invention is to provide a nucleotide sequence and its application. Using this nucleotide sequence, the present invention has also prepared primers, probes and kits that can detect Macrobrachium rosenbergii Dicistrovirus III, laying a reliable research foundation for detecting Macrobrachium rosenbergii Dicistrovirus III and studying the infection route of Macrobrachium rosenbergii Dicistrovirus III, etc.

[0004] To achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0005] In a first aspect, the present invention provides a nucleotide sequence, which is shown as SEQ ID NO.1.

[0006] In a second aspect, the present invention provides a protein, which has an amino acid sequence shown as SEQ ID NO.2 and encodes the ORF at positions 934 - 5808 in the nucleotide sequence shown as SEQ ID NO.1.

[0007] In a third aspect, the present invention further provides another protein, which is characterized in that it has an amino acid sequence shown as SEQ ID NO.3 and encodes the ORF at positions 5886 - 8609 in the nucleotide sequence shown as SEQ ID NO.1.

[0008] In a fourth aspect, the present invention provides primers for detecting the nucleotide sequence, which include an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are shown as SEQ ID NO.6 and SEQ ID NO.7 respectively.

[0009] In a fifth aspect, the present invention provides a probe that matches the primers for detecting the nucleotide sequence, and the probe sequence is shown as SEQ ID NO.8.

[0010] Preferably, the fluorescence quenching group of the probe is BHQ - 1, and the fluorescence reporting group is FAM.

[0011] In a sixth aspect, the present invention provides the use of the primers and / or the probe in the preparation of the nucleotide detection product.

[0012] Preferably, the nucleotide detection product includes a kit and a test strip.

[0013] In a seventh aspect, the present invention provides a nucleotide fluorescence quantitative PCR detection product, which includes the nucleotide sequence, the primers, and the probe.

[0014] In an eighth aspect, the present invention provides a method for differentiating the nucleotide sequence for non - diagnostic purposes, including:

[0015] (1) Configure the qPCR reaction systems for the negative control, positive control, and the sample to be detected. Among them, the primers and probes used in the qPCR reaction system are the primers shown as SEQ ID NO.6 and SEQ ID NO.7 and the probe shown as SEQ ID NO.8;

[0016] (2) Amplification of qPCR reaction system;

[0017] (3) According to the amplification curve of the qPCR reaction, determine the nucleotide sequence in the sample to be tested. The determination method is as follows:

[0018] If there is an amplification curve and the CT value ≤ 35, it is determined as positive for the nucleotide sequence; if the CT value is greater than 35, it is determined as negative for the nucleotide sequence.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention obtains a nucleotide sequence, specifically the complete genome sequence of Macrobrachium rosenbergii dicistrovirus type III. Through this sequence, the gene structure and function related to the proliferation and important regulatory functions of Macrobrachium rosenbergii dicistrovirus type III can be understood, the molecular physiological mechanism and the key points of how it infects the host can be found, and the virus pathogenic or even lethal genes can be searched. In addition, this nucleotide sequence is crucial for the design of primers, probes, and kits for the detection of Macrobrachium rosenbergii dicistrovirus type III, and also has important guiding significance for the subsequent development of related drug products. Brief Description of the Drawings

[0021] Figure 1 It is the prediction of the three-dimensional structure of the MrDV-3 capsid protein.

[0022] Figure 2 It is the result of sequence alignment of the capsid protein binding region.

[0023] Figure 3 It is the detection result of different dilutions of the MrDV-3 virus solution. Detailed Embodiments

[0024] The following further describes the present invention in detail with reference to the drawings and specific embodiments, which are explanations of the present invention rather than limitations.

[0025] Example 1

[0026] 1) Extraction of viral RNA

[0027] Take the hepatopancreas of Macrobrachium rosenbergii infected with Macrobrachium rosenbergii dicistrovirus type III CN-20220118 strain in Huzhou City, Zhejiang Province, and perform tissue grinding. Add PBS buffer at a volume ratio of 1:10, and ultrasonically break for 3 - 5 seconds to break the cells. After centrifuging at 6000 rpm for 30 min at 4°C, take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and then perform sucrose / glycerol discontinuous density gradient centrifugation to obtain high-purity MRDV-3 virus, and extract viral RNA using a viral RNA extraction kit.

[0028] 2) Reverse transcription PCR (RT-PCR)

[0029] Design and synthesize primer 1: 5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3' (SEQ ID NO.4), where N represents degenerate bases, including 4 6 possibilities. Take 5 μL of the previously prepared RNA virus and place it in a 200 μL special PCR amplification tube. Add 100 pmol of primer 1, place it at 65 °C for denaturation for 10 min, then immediately place it on ice. Add 2 μL of 10× reverse transcription buffer, 1 mmol / L 4× dNTP, 20 U RNasin, and 50 U Expand Reverse Transcriptase, and add water to a final volume of 20 μL. After mixing, incubate at 42 °C for 1 h.

[0030] 3) PCR amplification

[0031] Design and synthesize the following primer 2.

[0032] Primer 2: 5’-GCCGGAGCTCTGCAGAATTC-3' (SEQ ID NO.5).

[0033] Take 5 μL of the above reverse transcription product and place it in a 200 μL special PCR amplification tube. Add 2 μL of 10× PCR buffer, 350 nmol 4× dNTP, 700 nmol primer 2, 8 U Taq DNA polymerase containing Deep Vent, mix well and place it in a PCR instrument for amplification. The cycling parameters are: 95 °C for 3 min for 1 cycle, 94 °C for 30 s, 54 °C for 30 s, 68 °C for 3 min for 40 cycles. Take 50 μL of the reaction product for detection by 1% agarose gel electrophoresis, and cut and recover and purify the fragment with a size of 1 - 2 Kb.

[0034] 4) Ligation reaction

[0035] Perform ligation with the pMD20-T plasmid under the conditions shown in Table 1 below to obtain the ligation reaction product.

[0036] Table 1 Composition of the reaction system

[0037] 10× Ligation Buffer 1 μL DNA 7.5 μL pMD20-T 0.5 μL T4 Ligase 1 μL

[0038] 5) Transformation

[0039] Take 50 μL of Escherichia coli DH5α competent cells, add the ligation reaction product, place it on ice for 30 minutes. Place it at 42 °C for 1 minute, then perform an ice bath for 2 minutes. Add 500 μL of SOC nutrient solution at 37 °C, shake the bacteria at 37 °C for one hour, and coat the plate.

[0040] 6) Sequencing

[0041] Then, the positive clones of Escherichia coli were selected and PCR amplified using the M13 primer set (M13F: CGCCAGGGTTTTCCCAGTCACGAC, SEQ ID NO.23; M13R: CAGGAAACAGCTATGACC, SEQ ID NO.24), and the amplification conditions were: 95°C for 5 min, 95°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec for 35 cycles, and 72°C for 5 min. The PCR products were sent to a sequencing company for terminal dideoxy sequencing, the obtained sequences were spliced and aligned, and the PCR method was used to fill in the gaps, and the viral terminal sequences were obtained by RACE method (RACE amplification kit of TAKARA).

[0042] After repeated sequencing, on the basis of sequencing each base about 6 times on average, the complete genome sequence of Macrobrachium rosenbergii dicistrovirus type III was obtained as shown in SEQ ID NO.1.

[0043] SEQ ID NO.1:

[0044] TCCGATCGATTTACTGTCCTGTACATTTATGTTTCGCGCTCCTCTCGGGGGGCATTCGGG

[0045] GTCAACTTTGTTGACCCCGTTAGGGTGGAGATTTTATCTCTGCCTGCCTTTTGTTGGTTTT

[0046] AGCACCCAACTACAATTCGGAGATTGTGCCGTTGTTTCATCTTTTAGGAAGCGTAGCGG

[0047] CGCAGTAAGTGCACCGGCGTAAATCCTATCATTTTGAATACACTTCACTTTATCAAGTC

[0048] TCATATTTCCAACGAGATTGGCGGTGATGTACCAGTTCGCCGGATCCACGTAGTTATTG

[0049] TGAGAAGAGAATCTCGAAGCTGTAATTGCTTGATTGTGATAATTCACTACCCCCCTCCC

[0050] CCATGATGGTATTCCATCACAACAACACTTATTTATTGGTGTGGCATTTTCTGCTCACCT

[0051] TCAATTTAGACACATTCGATGCGTTTTTAGCGCTTTGCACCTAGTCCCTTGTGGTGCGGT

[0052] GTAGGTCTTTTAATTGTAAATTTATGTTTTATTTGTTTGTAAATTAGCTCTTGTATGGAG

[0053] CCAAAATAAAAATTTGTTTTTACTTTTATGAAAAATTAGTCCACTTAAGATTTCGTTAG

[0054] CTTTGCACGTTAGTATCTTAGTCATTATTCCACGTTTAGGAGTGGCCCCTAGATCTCTAT

[0055] TCCGAGTACCCGCTGTAGACCTCTTTGGAGGTTCGGCTTTTAGGATTGATCGCCTTGTG

[0056] CCACATTATCTTTTGACGGAGAACGCTTATCTGAGGCGAATATTTTTATTTGGAAGTCT

[0057] GGAATCTCTTTATCCCGCTTGGCAGCAGGGAACTGTAGTTTTCTTTGGCGTTTAATTTTT

[0058] CTATATATATTGTTTTCTGTTGTACTTTATTTGCACTGAACCACAAAAATATAAATATGC

[0059] GTAGGAGTCTTAGCGACGCTCCACCCCTTTTTCTGAAACTATGAAGTCGATTATTAATA

[0060] ACATCGACCCCAAGTCTGCCACAGAAACCCAAGTTAGCGCTGATGCGCACCAATCTTT

[0061] CGCTCTATTGTCACGAGCACTTGCTCGTAATGATATGAAAATCATTAACGTTGATGAGC

[0062] GTACTCGGAGACTTTATTTCGATGTCTCCATTCTTGACCGGAAATATCGTATTTTTGGTC

[0063] ATCATCGTGGAGAGGATATCTATAGGAGTATTCAAGTATCCATTAAGGATACAATATA

[0064] TGGATCTCTAAGATTCGCCGTCATGAACGGTCCTCTCTTCGTTCCCAACACTCCTGATA

[0065] GTTTAATTAGTATCTCCTTAATTGGGGAAACGTTAAACGTTACATTTGATAGCATTGGT

[0066] AATGACGCCTCTGTTCTATCCTGGAGTGTTCTTTTTGAACGAGCTTGTATGAACGCAGA

[0067] TATGCGTGTGCAAGCTGGTGAAGAAGATCCCGAAGATTTTGCTGCTTCTGCTGGTGGTA

[0068] TGGCCTTCATACTCATGCTTAAAATTGTTAGCAAGCAGCTCGCAGCACTCAACATCCCT

[0069] GATGCTAAGTTTTGGTTGGATATGTTTAATAATGTAGCCATTGCTTATCGAAGTTTCAA

[0070] GAGATGTGTATCCCTACAAGATTATGTGGACGAGATCCAAAAGTTTTACCGCATTTTCT

[0071] TAGGACGCAGCGCTTATTTAGACTGTTGCTCATACCTTGATGCTGTCATTGCCGACATT

[0072] TTCAAAGAGTCCACAGTCCAAGCTGATTCTACAGAAATGCTCCAGAATCTCCGCCAAG

[0073] CCTTTGATATGGTCACTGGTATTGGGGAGAATCCTACTGTTAAGAAGTTGCAATCTTTG

[0074] TTTTCATATGCACTTGTGCAGGGTTACTTAAAGCACTTCAACATGGAACTGAGTGATGA

[0075] AGACTATTCCAAAATGGAACAACGTCAATTGCTTAGTGCATATTCGTCCAAGCGAGGA

[0076] TTCTTTTTCGCTGTATTGGACACCACCCTCCATATTGCCGAGCGTTTAAATGCTTGGTAT

[0077] GTCACTGGTGATTTTGATAATTTCTTACATTCTGAGAAATTATATGTTGATTGGTTGAA

[0078] AGAAGCCGATAGACTGCTTGGTCTGGCTGCCTTTACATCAAATCTCACCGCACTTGGTG

[0079] ATGATAGCTTTAAGTTCAAGGCTGATTTGGAGGACGCTCTTGCTAAGGGAAGAGCGTA

[0080] CGCTAAGTTCACATCAAAGAGTGGTGGTACAGATTGTGTTGCCATTCGTAAGAAATTGT

[0081] GTGCTCTAGAATTACTCAATAATACTGAGATTACAAAGCGTGCTGCTCAAGTCGAACG

[0082] TAAAGCACCATTTGGTGTCTTGATTGCAGGTCATTCTAGTATTGCTAAATCAGCATTTA

[0083] CTAAAGTGATTTACAATGCTTATGGTTCTTTGTTTAATTTGGATCGATCTGATGCTGGTT

[0084] GTTATTGCCGTAACTCCTTTGATGAATTTTGGAGTGGATTTAATTCCTCACAATGGTGC

[0085] ATTCGAATGGATGATATTGCTTTCCAAAACCCAGCTAAGACCACGCAGATCGATAATA

[0086] GTATAATCGAAATGTTGAATATTGTCAATAACGTTCCGTTTGTCCCAAACCAAGCATCA

[0087] TTAGAGGCTAAGGGTACGACTCCTGTTCATGCTAAATTGGTTATTGCAACTACTAATAC

[0088] GTTGCATTTGAATACCAACGAATATTTTTCATGTCCCCTTGCTGCCAATCGGCGCCTTCC

[0089] TTATGTCATTGAATTATCACCTAAGAAGGAGCATCTACATGCTAACCAACTCTTTATCG

[0090] ATCCAACTACCCTTAAAAGTGAGGAAGGTAAGTTTCCTAATTTTTGGCATATAGTTGTA

[0091] AAGGAGATTGTTCCTGAGATCCGCACTGATGGAAAGGAGTATGCCAAGTATAAGGAAA

[0092] TCGCTGTGTTCGATGAGATTGATGATTTTATCGCACACTTTCTTAAGATGGCTCTCGTAC

[0093] ATGAAAATAATCAGGAACGTGCTGTAGCAAAGAACACTGATATGGCAACCATTGAGAT

[0094] TTGTAAGGTTTGTTTAAAACCTTTACCTCATAGTGGGTGTATGGATGTTCAGGCTGGAG

[0095] AATTGAGCATCATTGAGACCTATTTGGTTGCAGCGCTCATGTGGTTATTCAGTCTTAAG

[0096] TGGTTTGTCGAGGTAATTGTTCACCAATTATCTCGACGAGCTATGACTCGTTACCCTGT

[0097] TTACGCAGCAATCAATTATTTACCCCCGGAGGAAAGTGTTAGAATGTATGCTAGATTA

[0098] GCCGCACTACGACAGAATGTAAAAATTCAGCGACTTGTAGCAGGCCTCACTATCGTGT

[0099] CGGGAGCGTTTGCTCTTTATTATGCTACTCAAGGTAAAAAATTGAAGGCTCCTGTTCCT

[0100] CCTACTAATACTCAAACTTCTCATACGTATGAGAAAGTTAAAGAAGTATATGCTGAGG

[0101] AGAGTGTGGAAGATGTTCTCGAGCATGATGATCCTCTTGGTGTACAAGCTGATGGACG

[0102] ACTTGTTGAGACGCAGTTAGAGAAAGAGACAAAGAATAACGTTTGGTATAAAGCTGAC

[0103] GTTACTATCTCAACCTTTGACATGCCCACAGCATCCATGAGTTTGGCTAAAGCTACAGA

[0104] TGCCGAAGTTCGAAACATTCTAGACAAAAATTGCGTTGCAGTTGCAGTAAAGGCTGGT

[0105] GGTCATACCATCACTTTACGTGGAGTGTTTATAGTTGGACAAAAATTGTTGTTGCCAGC

[0106] CCATGCTTTTAAGTACATGACGAATGAATGCGAAATTAATGTTATTGATTCGGATATTG

[0107] CTAATTCCCACAACTCAAATTGTAAGTTTACACTTACAAAGAGCCAGTTGGTGATGATG

[0108] CCATCTATGGATTTATGTATGATTGAAGTAGCTGGTTTACCACCAAAAAAGAGCATTCT

[0109] GAAGTACTTTCTTCGCGAAGAGGTTTGTCCTACAAGGGGCTTTGAACTTATGCGTCAAG

[0110] AAGATGGTACTTTAGATATCATTCCCTTTTTCAATTTAAGGAAGGAGAGTGGGATGCCT

[0111] GTAGAATCTCTTGGAATCAAGGTAGATATTTACATGGGTATCAGTTCACAAGCTACAG

[0112] CTGCTGGAATGTGTGGATCTCTGTGTATCGGTTCCACTCCACGTGGACCAGTGATCATG

[0113] GGATTCCATCTCCTAGGTAATGGAAATCACGTTGGTTTCTTGTGTGTTAAAGCGCCTCA

[0114] AATTGAAGCACTTATGCAACACGAGAGTTTCCATCGTGTAGATGTTGAAGGTGGAGGT

[0115] ACACCAATGTTGCGTTGCTCTAAGCGAGCTTATGCAGTACAGTCCCTTCATCATCGTAG

[0116] TTTATTTCGCTACCTTCCTGCTGTGAATGCTAATTTGTATGGAACTTTAGATAGTTTTGC

[0117] TGGTAAGCAGAAATCTAAAGTTTGTGCCACACCGCTTCAAGCTGAAATTGTTCAAAAG

[0118] TATGCTCGTGACATTATGCATGGTGCTCCTGTTTTGGGAGGTTATCATGGTGTGAAACA

[0119] GAATGTTGCTCCTATGGTAGTTCGTACGAACAATTATAATAAGCTGTTTCTTAAAGATT

[0120] GTGTGAAAAGTTACTCCAATGACATCATTTCTCGTCTTAGTGAGCAAAGTAAGAAGGA

[0121] ATTAATTCCTCTGTCTTTGCATGCGGCTATTAATGGTTTACCTGGTGTGCGTTACATTGA

[0122] TGGTATTAATCGTAATACATCCATGGGATTTCCTTACAATACTACTAAGAAGGAATTTT

[0123] TAGAACCATGTGTTAGTGAGATGTATCCTGATGGTGTAACTTTCAAGCAGGAAGTGCTT

[0124] GAAGAGGTTAAAGCCATTGAAGCTACGTATGCTGAAGGTAGACGTGCGTACCCAATAT

[0125] TTGCAGGTCATAATAAGGATGAAGCCGTCACACTAGAAAAGGTGAAAGCTAAGAAGT

[0126] GTAGGTTATTTACTGGTTCACCTATTGCATGGAGTTTGGTAGTGCGAAAGCAATTACTC

[0127] ACCTTTGTGCGATGTGTACAGAAAAATCAGTTGATCTTCGAAGCTGGACCTGGATTAGT

[0128] TTGCCAATCTAATGAATGGAGTCTTGTGAGAGACCATTTATGTAAGTTCGGTGAAGATC

[0129] GAATTGTTGCTGGTGATTATGGAAAATTCGATAAACGTATGTTACCAGACTTTATTCTT

[0130] GCAGCGTTTGATGTTATTATAAATGTTTTGCGCGATGCGGGTTGGTCTGAACAAGAATT

[0131] ATTGGTAATTCAATGCATTGCATACGATATTGCTTTTCCGGTGTGTAGCATTAATGGTG

[0132] ACTTGGTTGAGTTCTTTGGAACTAATCCATCTGGTCATCCTCTTACAGTGATTGTTAATT

[0133] CCTTGGTGAACAGTTTGTATATGCGGTATTGTTATCGTGCTATGAATCCTGAACAAATT

[0134] GTTTCTGATTTCCAGGACCATGTTGCCCTATTTACATACGGTGACGACAACACTTTAGG

[0135] TGTGAGTAAACTTCGGAACTGGTATAACCACACAACGATTCAAGCTTGTTTGGCTCACA

[0136] TAGGCGTTGAGTATACGATGGCTGATAAGCTGTCAGAAAGTGTACCATACATCAATAT

[0137] TAGTGAAACATCTTTCCTAAAGCGTAAATGGCGATGGGATGAACGCATGCAATGTTAC

[0138] TTATGTCCTCTTGAGGAGGAATCTATTTTTAAGTCACTTACTGTATGGGTCCCTTCTGAT

[0139] ACTCTTGATAAATATTCACAATTCGTTAGGGTTGTTGAAAGTGCTGTTCAAGAATATTT

[0140] CTTTTACGGAGAGGAAAAGTTTGAAGAGATGAGGAACTACTTTATGAGTTTGCTTAGT

[0141] CAGGAACCTTATTCCTTGTATGTCACCAAATCCACATTTCCTACTTATGATGTACTTGA

[0142] GGCTCGATTTAAAGAAGCTTCTTCTGGCATGTAGGTTCAGGGAGTAGTGGATGTTTGCG

[0143] TGTGGTATTGTATATTGCATCACTTTTATAGATTCTGAATAGAATCTTACTTATGCGTTT

[0144] CCATTTGTACGTAATAAAAACATCTAAAATTAAGCCCCCCTGTGGGCTTGTGACTCGGA

[0145] AAGTCACACCTTCCCTTGCGTTAAGCCACGCGGGGAATTGTATCTACTGGTTGTTCAAA

[0146] ATTTATCTTATATGTTGTAAAGATGTTGATGTGGTTGCCAGAAATACCACAGCTCAAGG

[0147] AGAGTGTCGCTCAGCTCCCAAGAGTAAATATAGTGAGTACAAAGTCCAGTCTAGAGAA

[0148] TCTAGTGATACTACCAGTGAGGTATTAACATTTGTTGATAACTCAATAGGAGATGAGG

[0149] AAAAGGTGCAGTATGTGCCTAACCCTATCGCTTCTGCTGACGCAACATCAAACACTGA

[0150] TTTGGCCCGATTTTTAAGTCGACCAACACTGATTGATTCTCGTGGGTGGACCACAGCCA

[0151] ACTCAGTTGGTTATTTGGGTGCTGGAATAGAACCCTGGTATTTATACCTAAATAATGGT

[0152] GTTATTAAACAAAAATTGACTAATTATGCTTATTTACGAGCTAAATTATGTGTGAAATT

[0153] TGTTGTAAACGCTACACCTTTCCATTTTGGATGTTTGCGAGTCGCTTATGAACCCAATA

[0154] CTAACGTAGCCAATACAGGCTCACGTAGTTCTATGATTCGTACTAATCCTACATCAGAC

[0155] AATCCCTTGTTGATCCCATTGTCACAACTGCCAGGTGTGTGGCTCCATCCTGCTGATAA

[0156] TTCGGGAGGATATTTGGAGTTACCATTCTTCAAGGCTACAAATTGGTTATCACTGCAAA

[0157] CAGCGGCGGAAGCCAAAACCATGGGAGTTTTGAAGTATTTTATTGCCACCGTTTTAGGT

[0158] GCTGCGAGTGCTTCAGCTTCAACCGCGATTACTATCGACACATTCGCGTGGTTGGAGGA

[0159] TGTTGAGTTGAATGCCGCGACGGCGGAACTCACATTGCAAGGTAAGGATGAATATGAT

[0160] GGTCCCGTATCAAGCGTGGCATCAGCTGTTGCTTCTGTGTCAAAGCGACTGGAGACAG

[0161] CACCAGTTATTGGTAAGTTTGCTCGTGCGACGACCATTGGTGCTGGGGCTATAGCAGAT

[0162] ATCGCTAGTATGTTTGGTTTCACAAATGTACCAGTAATTGATAGTTATAAGCCTATGAT

[0163] GAATATGGCAGCGCCGCCTTTGGCTACTGGTGAGATTGGGGCTCCAATCGTAAAATTA

[0164] ACACTAGATCCGAAACAGGAACTGTCAGTGGATCCCTCTCTCCACGGGATCGGAAGTG

[0165] AAGATGAGATGGCTATTTCAACAATCGCGCAAAAATCTAGTGTTCTAGCTGTAACTGG

[0166] TTGGAGCACTACTGACACTATAGGAACCGTGTTGTTTACATCACGTGTTTCCCCCATGT

[0167] TAATGGGTCGTCAGGATATTTACGATGCAGGATCAGTTGCCCGCTCCACTCGTGTGTAT

[0168] CACACTCCCATGTCTTATTTGGGGATGTTGTTTATGCATTGGCGGGGCGATATCATCTA

[0169] TGACTTCGAAGTGATATGCACAAAATTCCATAAAGGGCGTATTAAGATCTCTTGGGAC

[0170] CCAGTGGGTACCGCTGGTGGAGCTGCTCTACCAGAAAATGTAGTTTATACAACCATTTT

[0171] GGATATCGGTGAATCCAATAAGGCTTCACTGCGTGTGCCATTTCACAGTGCCTATGCAT

[0172] TTTGTCGAATGCGAGGTATAGCGGCTGATAATTGGAATCCGGGTGATCCACTACCAAG

[0173] TGATCCTAAATTTGATAATGGTCTTTTGAATGTAGCAGTTCTTACACCACTGATTTCACC

[0174] AGTATCACCGCAAAATTTGGGTATTATTATTACCGTACGTGCGGCTCCAAATTTGGAGT

[0175] TTGCGAACCTGCGTAGTAGTCTTGCAGAAAATGATGGTACTCCACCACCTTCTTTCTTC

[0176] GCCGTTCAAGCAAAGGATGACGTTGATATTGAATCTAAAGAAGAGACTTTTGGAGACA

[0177] CTGGTTCACAGCACCCTCAACGTTACGCTTTGAATTTCGGAGAGTGCATTTCCTCATTG

[0178] CGAAGTGTTGCTCATCGTATGTCATTATATGACGTTAGTGCTCCTGGACCTAATGCGGC

[0179] GACGCGTTTTTTGTTAGCACGAAAGTCTTATTCTCGACTACCACCTATGTATGGTTATG

[0180] ATCCTAATGGAAGGAGTTCGGCCACTAGGGTTTTAGCAGGTACCGGTGCCGCAGCATT

[0181] CAACTTTACACCCACGCATCCTATGACTTATATAGCCATGATGTATGGTGCCTTCCGTG

[0182] GTAGTACAAACTACACCGCCTCTCCCGCTACGGATTTATATCCGTATATTGGAGATGTA

[0183] AGAGTTCAACGGCATACGCATGGAACATATGGTACTGCGCGTAGAGCACAATGGTTAA

[0184] CAACTCTTAATACAGGTGCTGCAGGTAGTGTAGCAGCGGAATGGTTGAATAATTGTCA

[0185] GGCCCTAACTGGGGGTGGCACAATGACTAATACTCAAGCGGGTGGACCAATTTCTTGG

[0186] AACGCCCCACACATGGGACCCACAAATTTTAATTTTTGTGACCCTACATACCTGAATGT

[0187] TGGAAATCCGACAGATCAGACTGATCTCGAATGTACTACTCTAGAGATTTTGATGCACC

[0188] AAACAACAGCTAATACTGTGTCTGATCAATTGGCTATCACTACCTATGCAGGTAGTGGT

[0189] GTCGATTGGCACTGTGTGTGGTGGTTGGCGTGTCCAACTTTGGATTATTACCTCACGAG

[0190] ACCCACAGGTGTTTAACCACCTTCTTTCTGTACCTTGAGGTGCAAGGGCGGCTTTAATT

[0191] AAAAAATACCTCTTGACGAGGACCGCAGAAAAGCTACGGCTTGATATTAAAGGGCTAG

[0192] CACATGCCACGACGCGAGGTGTGTTAGTCATAAATGGTAAAGCGCAAAGAAAAACGG

[0193] GATGTTGCAGTCTACCCCGTTCTCCTGCATTAGTGGAGTTTTAAAACATGGCCCCTTAG

[0194] GTCAAAGATTATCGTGATGGATGTAAGTCCTCGGTCTTTGGCCGGGGAAAGAACCATG

[0195] AACGAGTTGCAGTCTTTCGATCCGTATGGAGCTGTGCAGGAATTGTAC

[0196] The ORF at positions 934 - 5808 of SEQ ID NO.1 encodes a viral protease and RNA polymerase pre - proprotein with a length of 1624 amino acids (the amino acid sequence is shown in SEQ ID NO.2), and the ORF at positions 5886 - 8609 of SEQ ID NO.1 encodes a viral capsid pre - proprotein with a length of 907 amino acids (the amino acid sequence is shown in SEQ ID NO.3).

[0197] SEQ ID NO.2:

[0198] MKSIINNIDPKSATETQVSADAHQSFALLSRALARNDMKIINVDERTRRLYFDVSILDRKYRI

[0199] FGHHRGEDIYRSIQVSIKDTIYGSLRFAVMNGPLFVPNTPDSLISISLIGETLNVTFDSIGNDAS

[0200] VLSWSVLFERACMNADMRVQAGEEDPEDFAASAGGMAFILMLKIVSKQLAALNIPDAKF

[0201] WLDMFNNVAIAYRSFKRCVSLQDYVDEIQKFYRIFLGRSAYLDCCSYLDAVIADIFKESTV

[0202] QADSTEMLQNLRQAFDMVTGIGENPTVKKLQSLFSYALVQGYLKHFNMELSDEDYSKME

[0203] QRQLLSAYSSKRGFFFAVLDTTLHIAERLNAWYVTGDFDNFLHSEKLYVDWLKEADRLLG

[0204] LAAFTSNLTALGDDSFKFKADLEDALAKGRAYAKFTSKSGGTDCVAIRKKLCALELLNNT

[0205] EITKRAAQVERKAPFGVLIAGHSSIAKSAFTKVIYNAYGSLFNLDRSDAGCYCRNSFDEFWS

[0206] GFNSSQWCIRMDDIAFQNPAKTTQIDNSIIEMLNIVNNVPFVPNQASLEAKGTTPVHAKLVI

[0207] ATTNTLHLNTNEYFSCPLAANRRLPYVIELSPKKEHLHANQLFIDPTTLKSEEGKFPNFWHI

[0208] VVKEIVPEIRTDGKEYAKYKEIAVFDEIDDFIAHFLKMALVHENNQERAVAKNTDMATIEIC

[0209] KVCLKPLPHSGCMDVQAGELSIIETYLVAALMWLFSLKWFVEVIVHQLSRRAMTRYPVYA

[0210] AINYLPPEESVRMYARLAALRQNVKIQRLVAGLTIVSGAFALYYATQGKKLKAPVPPTNTQ

[0211] TSHTYEKVKEVYAEESVEDVLEHDDPLGVQADGRLVETQLEKETKNNVWYKADVTISTF

[0212] DMPTASMSLAKATDAEVRNILDKNCVAVAVKAGGHTITLRGVFIVGQKLLLPAHAFKYM

[0213] TNECEINVIDSDIANSHNSNCKFTLTKSQLVMMPSMDLCMIEVAGLPPKKSILKYFLREEVC

[0214] PTRGFELMRQEDGTLDIIPFFNLRKESGMPVESLGIKVDIYMGISSQATAAGMCGSLCIGSTP

[0215] RGPVIMGFHLLGNGNHVGFLCVKAPQIEALMQHESFHRVDVEGGGTPMLRCSKRAYAVQ

[0216] SLHHRSLFRYLPAVNANLYGTLDSFAGKQKSKVCATPLQAEIVQKYARDIMHGAPVLGGY

[0217] HGVKQNVAPMVVRTNNYNKLFLKDCVKSYSNDIISRLSEQSKKELIPLSLHAAINGLPGVR

[0218] YIDGINRNTSMGFPYNTTKKEFLEPCVSEMYPDGVTFKQEVLEEVKAIEATYAEGRRAYPIF

[0219] AGHNKDEAVTLEKVKAKKCRLFTGSPIAWSLVVRKQLLTFVRCVQKNQLIFEAGPGLVCQ

[0220] SNEWSLVRDHLCKFGEDRIVAGDYGKFDKRMLPDFILAAFDVIINVLRDAGWSEQELLVIQ

[0221] CIAYDIAFPVCSINGDLVEFFGTNPSGHPLTVIVNSLVNSLYMRYCYRAMNPEQIVSDFQDH

[0222] VALFTYGDDNTLGVSKLRNWYNHTTIQACLAHIGVEYTMADKLSESVPYINISETSFLKRK

[0223] WRWDERMQCYLCPLEEESIFKSLTVWVPSDTLDKYSQFVRVVESAVQEYFFYGEEKFEEM

[0224] RNYFMSLLSQEPYSLYVTKSTFPTYDVLEARFKEASSGM

[0225] SEQ ID NO.3:

[0226] MRFHLYVIKTSKIKPPCGLVTRKVTPSLALSHAGNCIYWLFKIYLICCKDVDVVARNTTAQ

[0227] GECRSAPKSKYSEYKVQSRESSDTTSEVLTFVDNSIGDEEKVQYVPNPIASADATSNTDLAR

[0228] FLSRPTLIDSRGWTTANSVGYLGAGIEPWYLYLNNGVIKQKLTNYAYLRAKLCVKFVVNA

[0229] TPFHFGCLRVAYEPNTNVANTGSRSSMIRTNPTSDNPLLIPLSQLPGVWLHPADNSGGYLEL

[0230] PFFKATNWLSLQTAAEAKTMGVLKYFIATVLGAASASASTAITIDTFAWLEDVELNAATAE

[0231] LTLQGKDEYDGPVSSVASAVASVSKRLETAPVIGKFARATTIGAGAIADIASMFGFTNVPVI

[0232] DSYKPMMNMAAPPLATGEIGAPIVKLTLDPKQELSVDPSLHGIGSEDEMAISTIAQKSSVLA

[0233] VTGWSTTDTIGTVLFTSRVSPMLMGRQDIYDAGSVARSTRVYHTPMSYLGMLFMHWRGD

[0234] IIYDFEVICTKFHKGRIKISWDPVGTAGGAALPENVVYTTILDIGESNKASLRVPFHSAYAFC

[0235] RMRGIAADNWNPGDPLPSDPKFDNGLLNVAVLTPLISPVSPQNLGIIITVRAAPNLEFANLR

[0236] SSLAENDGTPPPSFFAVQAKDDVDIESKEETFGDTGSQHPQRYALNFGECISSLRSVAHRMS

[0237] LYDVSAPGPNAATRFLLARKSYSRLPPMYGYDPNGRSSATRVLAGTGAAAFNFTPTHPMT

[0238] YIAMMYGAFRGSTNYTASPATDLYPYIGDVRVQRHTHGTYGTARRAQWLTTLNTGAAGS

[0239] VAAEWLNNCQALTGGGTMTNTQAGGPISWNAPHMGPTNFNFCDPTYLNVGNPTDQTDLE

[0240] CTTLEILMHQTTANTVSDQLAITTYAGSGVDWHCVWWLACPTLDYYLTRPTGV

[0241] Example 2

[0242] Verification of the genome sequence of Macrobrachium rosenbergii dicistrovirus type III

[0243] According to the nucleotide sequence shown in SEQ ID NO.1, primers were designed at a length of about 1400 bp each (see Table 2), and PCR reactions were carried out. The PCR products were sequenced and verified. The results showed that the nucleotide sequence of SEQ ID NO.1 was correct.

[0244] Table 2 Primers for amplifying the full sequence

[0245]

[0246] Example 3

[0247] Kit for detecting Macrobrachium rosenbergii dicistrovirus type III

[0248] When the Macrobrachium rosenbergii dicistrovirus type III (MrDV-3) enters cells, it first binds to receptors on the cell surface through the viral capsid protein. After fusing with the cell membrane, it enters the cell for replication. Therefore, the capsid protein plays a decisive role in determining the types of cells that the virus can infect. There are significant differences in the capsid proteins of different virus species, and for the same virus, the capsid protein also shows conservation. Identifying the conserved regions of the capsid protein and screening the nucleic acid sequences are the keys to determining the detection targets and designing primers and probes for this virus. This kit is based on the genomic sequence shown in SEQ ID NO.1. Through various molecular biology software, the functional regions of the capsid protein ( Figure 1 shows the predicted three-dimensional structure of the MrDV-3 capsid protein. The predicted host-binding region, that is, the protein marked by the spherical ball shown by the arrow in the figure, has high host conservation. Therefore, the gene sequence encoding this protein is selected as the primer design region), and through codon preference, the target region of the detection sequence is identified ( Figure 2 gives the sequence alignment results of the capsid protein binding region, indicating that this region has a certain degree of conservation among viruses of the same genus. Therefore, it is speculated that it also has high conservation in this virus species. Since there is only the virus information involved in this patent for this species of virus, the sequence information of this region is unique under the existing conditions), and the conserved region of the binding region is obtained. Primers and probes for detecting MrDV-3 are designed in the conserved region.

[0249] Designed PCR primers and probes:

[0250] Forward primer 1: 5’-GCAAGGTAAGGATGAATATG-3’, SEQ ID NO.6, and the sequence is from positions 6812 - 6831 in SEQ ID NO.1.

[0251] Reverse primer 2: 5’-AGGCTTATAACTATCAATTACTG-3’, SEQ ID NO.7, and the sequence is the complementary sequence of positions 6982 - 7004 in SEQ ID NO.1.

[0252] Probe 3: 5’-FAM-CCGTATCAAGCGTGGCATCAG-BHQ1-3’, SEQ ID NO.8, and the sequence is the complementary sequence of positions 6838 - 6858 in SEQ ID NO.1.

[0253] Prepare a kit for detecting Macrobrachium rosenbergii dicistrovirus type III (usable for 100 detections), and its components are shown in Table 3:

[0254] Table 3 Composition of the kit

[0255]

[0256]

[0257] The giant freshwater prawn (Macrobrachium rosenbergii) was infected with Macrobrachium rosenbergii dicistrovirus type III. The Macrobrachium rosenbergii dicistrovirus type III strain was obtained as in Example 1, and a cDNA sample was obtained by RT-PCR. The cDNA sample was diluted 10 -4 , 10 -5 , 10 -6 and 10 -7 times to prepare samples A, B, C, and D. PCR amplification and hybridization detection were performed using the above kit, and positive and negative controls were set up simultaneously.

[0258] The results are as Figure 3 shown, indicating that the kit can detect MRDV-3 samples ( -4 , 10 -5 , 10 -6 and 10 -7 times diluted). Figure 3 ).

[0259] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. A primer for detecting a viral nucleotide sequence, characterized in that, The primers include an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively; the viral nucleotide sequence is shown in SEQ ID NO.

1.

2. Primer-probe combinations for detecting viral nucleotide sequences, characterized in that, The primers are the primers described in claim 1, the probe sequence is shown in SEQ ID NO.8, and the viral nucleotide sequence is shown in SEQ ID NO.

1.

3. The primer-probe combination according to claim 2, wherein The fluorescence quenching group of the probe is BHQ-1, and the fluorescence reporting group is FAM.

4. Use of the primer described in claim 1 or the primer-probe combination described in claim 2 or 3 in the preparation of a kit for detecting a viral nucleotide sequence, wherein the viral nucleotide sequence is shown in SEQ ID NO.

1.

5. A nucleotide fluorescence quantitative PCR detection product, characterized in that, Comprising the viral nucleotide sequence shown in SEQ ID NO.1 and the primer-probe combination described in claim 2 or 3.

6. A method for identifying the viral nucleotide sequence shown in SEQ ID NO.1 for non-diagnostic and non-therapeutic purposes, characterized in that, Comprising: (1) Configure the qPCR reaction systems for the negative control, positive control, and the sample to be detected. Among them, the primers and probes used in the qPCR reaction system are: the primers shown in SEQ ID NO.6 and SEQ ID NO.7 and the probe shown in SEQ ID NO.8; (2) Amplify the qPCR reaction system; (3) According to the amplification curve of the qPCR reaction, determine the nucleotide sequence in the sample to be detected. The determination method is: If an amplification curve appears and the CT value ≤ 35, it is determined as positive for the nucleotide sequence, and if the CT value is greater than 35, it is determined as negative for the nucleotide sequence.

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