NotV1, a soybean mottle virus, and its PCR detection primers and methods
By designing a PCR method with specific detection primers and probes, the gap in virus detection for plants of the genus *Pseudodra* such as *Pseudodra* was filled, enabling rapid and accurate detection of NotV1 virus and protecting the growth of important medicinal raw materials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-29
- Publication Date
- 2026-07-31
AI Technical Summary
The lack of virus detection methods for plants in the genus *Pterocarya*, such as *Pterocarya stenoptera*, makes it difficult to detect new viral infections in a timely manner, affecting plant growth and the protection of medicinal raw materials.
Specific detection primers and probes were designed, and a PCR detection method was established based on the whole genome sequence of NotV1 (notmativirus 1) for rapid and accurate detection of the virus.
This study enriched the variety of viruses in the soybean mottle virus genus, expanded the host range, improved the detection capability of viral diseases in plants of the Cornaceae family, and protected sensitive plants and extremely small populations.
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Figure CN122484356A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of plant disease diagnosis and relates to a method for detecting pathogens in plants of the Cornaceae family. Background Technology
[0002] *Nothapodytes nimmoniana*, also known as crisp branch or stinky false notoginseng tree, is a plant belonging to the genus *Nothapodytes* in the family Cornaceae. *Nothapodytes nimmoniana* and other plants in the genus *Nothapodytes* are high-quality raw materials for extracting the anticancer drug camptothecin and its derivatives. Among all plants known to contain camptothecin, *Nothapodytes nimmoniana* has the highest camptothecin content. Due to the extremely high demand for camptothecin, the population of *Nothapodytes nimmoniana* is declining sharply and has been listed as a sensitive species in some countries. In my country, it is classified as a very small population. In recent years, there have been cases of artificial introduction, rapid propagation, and cultivation of *Nothapodytes nimmoniana* in Guangdong, Yunnan, and Fujian provinces of my country. The introduction and domestication of plants has significant production value, but the process is often extensive and lacks targeted inspection and quarantine measures, leading to the introduction of new viruses. Therefore, it is necessary to conduct virus testing on newly introduced *Nothapodytes nimmoniana* and other plants in the genus *Nothapodytes* to determine their growth status and pathogen-carrying status.
[0003] Soymovirus belongs to the family Caulimoviridae. The particles of this genus are spherical, 50 nm in diameter, and contain a single, non-covalently closed circular double-stranded DNA (dsDNA) genome. The genome contains 7-8 open reading frames (ORFs), and its genome composition is very different from that of closely related genera. The host range of the genus *Soybean Mottle Virus* is very narrow, with only seven known viruses in this genus: *Cestrum yellow leaf curling virus* (CmYLCV), *soybeanchlorotic mottle virus* (SbCMV), *peanut chlorotic streak virus* (PCSV), *water chestnut soymovirus 1* (WCSV1), *Malva-associated soymovirus 1* (MaSV1), *Hibiscus soymovirus* (HSV), and *Blueberry red ringspot virus* (BRRV). Summary of the Invention
[0004] In 2023, multiple *Nothapodytes* plants suspected of being infected with a viral disease were discovered at the Introduction and Domestication Garden of the Fujian Subtropical Plant Research Institute. Affected plants exhibited obvious mosaic, mottling, and curling symptoms on their leaves. Through high-throughput RNA-seq sequencing combined with bioinformatics, PCR, and sequence alignment, the pathogen was identified as a novel virus belonging to the genus *Soymovirus*. In the context of this application, this virus is referred to as *Nothapodytes* virus 1 (NotV1). After full-length amplification and sequencing, the genome of this novel virus is a circular dsDNA, 6671 bp in length, and its sequence is shown in SEQ ID NO. 1. Using ORF Finder software, the viral genome was predicted to contain 7 open reading frames (ORFs). ORF1 encodes a hypothetical protein of 31 amino acids, ORF2 encodes a 9.6 kDa protein (87 aa), ORF3 encodes a hypothetical polypeptide of 39 amino acids, ORF4 encodes a 44.4 kDa capsid protein (404 aa), ORF5 encodes a 71.9 kDa polymerase (reverse transcriptase + RNase H) (654 aa), ORF6 encodes a 16 kDa translation activator (145 aa), and ORF7 encodes a 35.8 kDa mobile protein (325 aa).
[0005] PCR is one of the most commonly used methods for virus detection, and it is a nucleic acid-level detection method. Compared with biological detection, electron microscopy, and serological detection methods, it has many advantages: high sensitivity, capable of rapidly amplifying extremely small amounts of nucleic acid to detectable levels, with sensitivity reaching the pg level; and high specificity, unaffected by extraneous proteins in the body.
[0006] NotV1, which infects *Pterocarya* var. *marginata*, is a novel DNA virus identified by the applicant that infects *Pterocarya* var. *marginata* and other plants in the *Pterocarya* genus. This virus affects the growth of these plants, and there are currently no reports on detection methods or primers for this virus. This invention designs five pairs of primers based on the virus's nucleotide sequence for specific detection, and establishes a PCR method for detecting NotV1 using these designed specific primers, providing an effective tool for the detection, diagnosis, and control of this virus.
[0007] Therefore, one of the objectives of this invention is to provide a PCR detection primer and method for a novel soybean mottle virus, Stinking Mabimu Virus No. 1.
[0008] A Soymovirus, wherein the genome of the virus is a covalently closed circular double-stranded DNA, and the virus contains the nucleotide shown in SEQ ID NO: 1 or a nucleotide that is completely complementary to the nucleotide shown in SEQ ID NO: 1.
[0009] An isolated nucleotide comprising a nucleotide selected from: the nucleotide or fragment thereof shown in sequence SEQ ID NO: 1; a nucleotide that is completely complementary to the nucleotide shown in sequence SEQ ID NO: 1.
[0010] A primer pair or probe selected from the nucleotides shown in sequences SEQ ID NO: 17 to SEQ ID NO: 26.
[0011] The use of the primers or probes in diagnosing plant virus infections, wherein the plant is one or more of the following species: Nothapodytes pittosporoides, Nothapodytes collina, Nothapodytes nimmoniana, Nothapodytes obscura, Nothapodytes obtusifolia, and Nothapodytes tomentosa.
[0012] Those skilled in the art understand that although a specific primer pair is used for the detection of newborn organisms in specific embodiments, this primer pair is merely exemplary and should not be construed as limiting the scope of protection of this application. Those skilled in the art, based on textbooks and the principle of nucleotide sequence complementarity (e.g., *Molecular Cloning: A Laboratory Manual*, J. Sambrook & MR. Green, 2017; p. 450 "Designing PCR Primers Using Primer3 Plus"; Chapter 13 "Preparation of Labeled DNA Probes, RNA Probes, and Oligonucleotide Probes"), are familiar with the principles of primer amplification of target sequences, the principles of probe binding to target sequences, and the design principles of primers and probes. Various primer / probe design software programs are available in the prior art, such as Primer Premier, Oligo7, and BeaconDesigner. When those skilled in the art know the genome sequence of an organism or its functional fragments (such as ORFs), they can obtain specific primer or probe sequence and structural information.
[0013] In a preferred embodiment of the present invention, organism-specific primers can be designed based on the following principles:
[0014] 1) The primers are 15 bp to 30 bp in length (including the endpoints, the same below);
[0015] 2) The G+C content in the primers is 40% to 60%;
[0016] 3) There cannot be four consecutive complementary bases within or between the primers;
[0017] 4) The Tm value of the primer is 55±5℃;
[0018] 5) The length of the amplification product obtained by the primers is 500 bp to 3000 bp (preferably 1000 bp to 2000 bp).
[0019] 6) The primers are complementary to nucleotides selected from the following:
[0020] The nucleotide or fragment thereof shown in SEQ ID NO: 1;
[0021] Nucleotides complementary to the nucleotide shown in SEQ ID NO: 1;
[0022] According to some embodiments, the uses of the primers or probes described herein are also provided. In some embodiments, the uses of the primers or probes of this application are provided in the diagnosis of viral diseases (such as mosaic virus and leaf curl) in plants of the genus *Pseudodra* (e.g., *Pseudodra pubescens*).
[0023] No infection of other plant families by this new organism has been found so far. However, the possibility of it infecting other plant families cannot be ruled out.
[0024] The use of the primers or probes in identifying pathogens, wherein the identification refers to: determining whether the pathogen to be tested is the isolated organism; or determining whether the isolated organism is present in the sample.
[0025] The use of the primers or probes in the preparation of a pathogen detection device, wherein the pathogen is the isolated organism; and the detection device is in the form of a reagent and / or a chip.
[0026] A detection device comprising the aforementioned primers or probes; the detection device is in the form of reagents and / or chips.
[0027] A method for detecting pathogens, comprising the steps of:
[0028] 1) Provide the sample to be tested; the sample to be tested is derived from plants of the genus *Pterocarya*.
[0029] 2) Obtain the nucleotides of the sample to be tested; the nucleotides are DNA;
[0030] 3) Contact the nucleotides of the sample to be tested or their amplification products with the aforementioned primers or probes;
[0031] 4) Determine whether the aforementioned isolated nucleotides are present.
[0032] The beneficial effects of this invention are as follows: The inventors have identified a novel pathogen of the genus *Soybean Mottle Virus* in plants of the Cornaceae family, which is reported for the first time. Since there is no specific detection method for this pathogen, this application designs specific detection primers / probes based on the pathogen's complete genome sequence and establishes a method for detecting this pathogen. This enriches the variety of viruses in the *Soybean Mottle Virus* genus, expands the host range of viruses in this genus, and also expands the detection range of virus-related diseases in important medicinal raw materials (such as *Begonia suffruticosa* of the Cornaceae family), which is beneficial for protecting sensitive plants and extremely small populations. Attached Figure Description
[0033] Figure 1 Symptoms of mosaic virus in *Begonia rotundifolia*.
[0034] Figure 2 The genome structure of NotV1, a novel virus of *NotV1*.
[0035] Figure 3 Phylogenetic trees were constructed based on the polymerase nucleotide sequences of NotV1 and 18 viruses most similar to NotV1. Phylogenetic analysis was performed using maximum likelihood statistics with MEGA X software. The bootstrap method (1000 replicates) was used to assess phylogeny, and the assessment numbers are shown below the branches.
[0036] Figure 4 Phylogenetic tree constructed based on the amino acid sequences of NotV1 and 18 viral polymerases most similar to NotV1.
[0037] Figure 5 Establishment of the NotV1 detection method. Lanes 1-5 represent the fragments amplified in *Pterocarya stenoptera* samples by primer pairs NotV1F / NotV1529R, NotV1273F / NotV2904R, NotV2754F / NotV4147R, NotV3966F / NotV5447R, and NotV5243F / NotV6671R, respectively; M: DNA molecular weight marker. Detailed Implementation
[0038] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.
[0039] Example 1. Identification and sequencing of pathogens
[0040] In 2023, the inventors discovered *Nothapodytes nimmoniana*, a plant exhibiting symptoms of mosaic mottle disease, in Fujian Province, China. Figure 1 ).
[0041] Total RNA was extracted from symptomatic leaves using the mirVana™ microRNA (miRNA) Isolation kit (Ambion, Thermo Fisher Scientific, Waltham, MA, United States). Total RNA was purified using the RNA Clean XP Kit (Beckman Coulter, Brea, CA, United States) and the RNase-Free DNase Set (QIAGEN GmbH, Hilden, Germany). Ribosomal RNA was removed using the RiboZero Magnetic Kit (Epicentre, Lucigen, Middleton, WI, United States), and cDNA libraries were constructed using the TruSeqRNA Sample Prep Kit (Illumina, San Diego, CA, United States). Paired-end sequencing was then performed on the Illumina HiSeq X platform.
[0042] Sequencing yielded 77,896,020 reads. After removing low-quality reads, initial assembly was performed using CLC GenomicsWorkbench 6.0.4 (Qiagen, Valencia, CA, United States), followed by reassembly using CAP3 software, resulting in 11,834 contigs. BLASTx was used to align these contigs with the NCBI non-redundant (Nr) database (E-value < 1e−5). The results showed 46 contigs ranging from 231 bp to 3411 bp with high similarity to several members of the soybean mottle virus genus. This indicates that *Solanum lyratum* plants exhibiting mosaic mottle symptoms carry a soybean mottle virus.
[0043] To identify the characteristics of soybean mottle virus in *Solanum lycopersicum*, the inventors designed viral genome amplification primers based on the contiguous sequences of the HTS and the end-to-end structural characteristics of circular DNA viral genomes (Table 1). A strategy combining full-length amplification and segmented overlapping amplification was employed, supplemented by nested PCR in key regions to enhance specificity and sensitivity. Seven overlapping fragments were amplified using Ex Taq (TaKaRa, Dalian, China). All obtained PCR fragments were cloned into the pMD19 vector (Takara) for bidirectional sequencing. Three positive clones were sequenced from each PCR fragment. The complete viral genome sequence was then obtained by splicing the sequences.
[0044] Table 1. Primer pairs used for NotV1 genome cloning
[0045] Note: F” / R” indicates nested primers
[0046] The genome sequence of the target virus is 6,671 nt in length (SEQ ID NO: 1), and it is a typical circular DNA virus. BLASTn analysis showed that the complete sequence of this virus has the highest similarity to several soybean mottle virus species, with a nucleotide similarity of 75.15%-75.25%. The inventors speculate that the identified virus is a new soybean mottle virus species, and have tentatively named it Nothapodytes virus 1 (NotV1) (GenBank accession number PZ295458, not previously published in this application).
[0047] Genome analysis of NotV1 (a type of soybean mottle virus) revealed that it possesses seven open reading frames (ORFs) and exhibits typical genome structure characteristics of the soybean mottle virus genus. The genome encodes seven proteins / peptides: ORF1 encodes a 31 kDa protein (nt: 590–686), ORF2 encodes an 87 kDa protein (nt: 909–1173), ORF3 encodes a 39 kDa protein (nt: 1172–1292), ORF1–ORF3 are all unknown proteins, ORF4 encodes a 404 kDa coat protein CP (nt: 1297–2512), ORF5 encodes a 654 kDa polymerase (reverse transcriptase + RNase H) (nt: 2605–4602), ORF6 encodes a 145 kDa translation activator (nt: 4587–5025), and ORF7 encodes a 325 kDa mobile protein (nt: 5482–6460). Figure 2 ).
[0048] Phylogenetic analysis was performed using maximum likelihood statistics (1000 bootstrap replicates) in MEGA 7 software. Sequence alignment was performed using ClustalW. Phylogenetic trees were generated based on the polymerase nucleotide and amino acid sequences of NotV1 and 18 viruses from 6 genera of the Caulimoviridae family that are most similar to NotV1. Apple mosaic virus (AMV) of the Ilarvirus genus was used as a distant relation control. Phylogenetic trees constructed based on the polymerase nucleotide sequences of the viruses show that NotV1 clusters with seven members of the genus Soymovirus, including Cestrum yellow leaf curling virus (CmYLCV), soybean chlorotic mottle virus (SbCMV), peanut chlorotic streak virus (PCSV), water chestnut soymovirus 1 (WCSV1), Malva-associated soymovirus 1 (MaSV1), Hibiscus soymovirus (HSV), and blueberry red ringspot virus (BRRV), indicating that NotV1 is closely related to the genus Soymovirus. The phylogenetic tree constructed based on amino acids is consistent with the nucleotide phylogenetic tree. NotV1 clusters with members of the soybean chlorotic mottle virus genus and with blueberry red ringspot virus (BRRV) on the same branch, indicating that NotV1 is most closely related to BRRV. Figure 3 ,4).
[0049] The International Committee on Taxonomy of Viruses (ICTV) uses the following threshold criteria for classifying the genus *Soybean Chlorotic Mottle Virus*: (1) host range; (2) a nucleotide sequence difference of more than 20%, meaning a nucleotide sequence similarity of less than 80% with the polymerases of other viruses within the genus is considered a new virus. The host range of viruses in the *Soybean Chlorotic Mottle Virus* genus is relatively narrow. This invention is the first to discover that viruses in this genus can infect plants of the *Pseudolarix* genus, such as *Pseudolarix*, thus expanding the host range of viruses in the *Soybean Chlorotic Mottle Virus* genus. Simultaneously, the nucleotide and amino acid sequences of the NotV1 polymerase were compared pairwise with those of seven members of the *Soybean Chlorotic Mottle Virus* genus. The results showed that the NotV1 polymerase nucleotide and amino acid sequences had the highest similarity to BRRV, at 71.22% and 68.18%, respectively (Table 3). The polymerase nucleotide sequence difference was 28.78%, clearly exceeding the threshold criteria for classifying the genus *Soybean Chlorotic Mottle Virus*. In conclusion, NotV1 can be identified as a new virus in the *Soybean Chlorotic Mottle Virus* genus.
[0050] Example 2. Establishment of a detection method for NotV1 (Not Virus 1) of Stinkhorn.
[0051] 1. Primer design:
[0052] Based on the NotV1 virus whole genome sequence, primers for NotV1 detection were designed using Primer Premier 6 software. The primer design sites included any fragment at any position in the NotV1 full-length genome sequence (SEQ ID NO: 1).
[0053] The primer design principles are:
[0054] 1) The primers specifically bind to any segment of the NotV1 genome;
[0055] 2) Primer length is 15 bp to 30 bp;
[0056] 3) The G+C content is between 40% and 60%;
[0057] 4) There cannot be four consecutive complementary bases within or between primers;
[0058] 5) The Tm value of the primers is 55±5°C;
[0059] 6) The length of the amplified product is 500 bp to 3000 bp.
[0060] Exemplary detection primers designed based on the above design principles include:
[0061] Table 2. Exemplary detection primers designed
[0063] 2. The detection method is as follows:
[0064] Using the above-mentioned *Pterocarya stenoptera* samples for high-throughput sequencing as materials, and taking a pair of amplified partial sequences NOTsoy5243F and NOTsoy6671R as examples, a detection method for NotV1 was established.
[0065] Take 0.1 g of *Begonia suffruticosa* leaves and grind them thoroughly in liquid nitrogen. Extract the leaf DNA using the TIANGEN Universal Genomic DNA Extraction Kit (DP605). Refer to the instruction manual for specific steps. Dissolve the extracted DNA in 30 μL of DEPC ddH2O and store at -80 ℃ for later use.
[0066] The concentration of DNA was determined using Nanodrop 2000. PCR was performed using 1000 to 2000 ng of DNA as a template. The PCR reaction system consisted of: 1 to 2 μL of DNA template, 1 μL of dNTPs (2.5 mmol / L each), 0.5 to 1 μM of forward and reverse primers, 0.25 μL of DNA polymerase (5 U / μL), 2.5 μL of 10× DNA polymerase buffer, and ddH2O to a final volume of 25 μL.
[0067] PCR reaction procedure: 94-95℃ pre-denaturation for 3 min; 94-95℃ denaturation for 30 s, 50-60℃ annealing for 30 s; 72℃ extension for 45 s to 3 min, 30 cycles; 72℃ final extension for 10 min. 5 μL of PCR product was subjected to 1% agarose gel electrophoresis.
[0068] 5 μl of PCR product was analyzed using a 1% agarose gel electrophoresis. The results are shown in the figure. Figure 5 Using five primer pairs—NotV1F / NotV1529R, NotV1273F / NotV2904R, NotV2754F / NotV4147R, NotV3966F / NotV5447R, and NotV5243F / NotV6671R—PCR amplified target fragments of approximately 1529 bp, 1631 bp, 1393 bp, 1481 bp, and 1429 bp in NotV1-infected *Pterocarya stenoptera* samples. Sequencing experiments further confirmed that the amplified bands were NotV1 fragments.
[0069] Table 3. Sequence homology between NotV1 and Soymovirus-associated viral polymerases
[0070] unit:%
[0071]
[0072] Sequence information
[0073] 6671bp
[0074] NotV1 genome (SEQ ID NO:1)
[0075] 1 CGGACCTGAC TTTACATTTC ATTATAATTT ATTAAAAGAT AAAGACGGAC CGGACTTGAA
[0076] 61 GTTACAATCA AATGTAACTT ATATTTATTT CAGTTACAGA CTCGAAAACG ACAAAGAAAA
[0077] 121 AGTTTGAAAA TGACAAAGTA AAAGTAAAGT TGATGCTGAC ATTTCTGCTG CTTTAGTCAT
[0078] 181 TTTCATGGGG CCTACATGTC TGATGCTGTT TCTTCGATCA TTACTTCCTC TGTAGAATCG
[0079] 241 TAACTACTGC TTTCTTCACA ATATTTGCAA CTGTGTCTTG GCACTCTTCT AATCTTTTTG
[0080] 301 CATATTTTCT TTTGAATATC TTCTGGGAGA CCTGGTATTT GATTGCTGAG AAGTATTTTT
[0081] 361 TCGAAATCCA TGAATACCTT CTCTTGTTCT GGACTGATAG TGGTTGATGA TTTTGAATAT
[0082] 421 ATCAAACAAT TCTCAGATTT ATAATTTACT CTGATATTTG CTGTCTGATT TTGCAAATGA
[0083] 481 AATACTAATC CTGAAAATAA AGTAGAAATT CTTTTTTCCA TCTTTTTATT ACGAGAAAAT
[0084] 541 AGAGACTTAA ATAAAAATT ATTAGCTAAA CATTGTTCAA TTACAATTTA TGAAACTACC
[0085] 601 ATTTGAAATC CTGGTTATAG CGAGAAGCGC AGCAAAAATC TGGTATCAGA GCCAAAGAAA
[0086] 661 GAGTTTAGAA AAAATCAAGA AGAATTAATT ATGGCCGAAA GAATTATAAG TGTAACTGAT
[0087] 721 ATGGTAGAAG AAATCAATAA TAATATAAAC AAATAAATGA AAGAATAAGC CAATTAGAAG
[0088] 781 AAATTTGGAT ACCTAAACAT ATTATATCTA GTAATAAACC TATTTTAGAA CAATACTGTT
[0089] 841 CTCAGAAAAA AGAATTATT TCTCATTTAT ATCATACTTT AAATCCTCAA CTAAATAATA
[0090] 901 TTACTGATAT GATAAGTTAC ATAATAAAAC ACATCAATAA AGAAAAAGAT AAAGAAATGA
[0091] 961 TAGAATATAT AGATCTCGCT ATAAATCCTA GTTATATAAA ACATTTAGAA AAACAAGTAA
[0092] 1021 AAGAATTATT AGAAGAAAAT AAAAAATTAA TAGAACAACG ATGTGTTACT ATTCAAGATC
[0093] 1081 TTAAAGAACA TTTTAAAATA AAAGTAAAAG AATCAAATTT ATTTCTAGAA GAAAATAAAT
[0094] 1141 CTAGTCAACC ATTTTGGTTC AAAAATAGAA CROWNING CROWNING
[0095] 1201 AAAAAATTATTACK AAAAAATAAA TGAAAAATTG EXPECTATIONSG.
[0096] 1261 GGAGATGAAT CAGAAGATAG TGACTTTGAG GTAAAAAATGA ATAAAAAAA AGAAAAAATG
[0097] 1321 REPEAT CTAGTTATTC AAAAAAAAAAG REPEAT WATERCATTC CATTATCAA
[0098] 1381 GAACAAGCAA GAGATAGATT TAAAAATCGA TGGAAAAGAG ATAGAAATTG GGAATTTCAA
[0099] 1441 TCSEQUENCY CTGATTTCA NATIONAL SEQUENCE GTSEARCH WINDOW
[0100] 1501 CGAGGAAAAT GGATAAACA AGGAACTCCT CAPTAIN ATCAACCTAT AATCAACCTAT
[0101] 1561 GTAGGTCCAG AAGGAATTT AAATTTAGAT TGTGTAAAAA ATGGAAAAA AGTTTAAAG
[0102] 1621 ASSISTANT CTAAAACAAGC TTTAATGATC ASSISTANT AAGGATTAA AAATATGGAC
[0103] 1681 CHANNEL SERIES TTTAAATACH AAAACTTCAG COMMUNICATIONGTTTT SEQUENCES
[0104] 1741 SEQUENCES SEQUENCES GCAAGAAAAT SEQUENCES SEQUENCES
[0105] 1801 GGAACAAAA TTTATCAAGA GTTCTTAGGA GGAATGAGTA CTCATATAAC TACAAAATCC
[0106] 1861 GATAGTTATG ATATAGAAG AATAAAATGG ATTTTAAATC AAATTACTTT ATGTAATATG
[0107] 1921 TGTGATTTTG STATETTGC ATGTCAATAT GROUP ATTAGTATT ACCAGTAACC
[0108] 1981 GAAATA TATTTCTAAT HELPTTCGTA AACAATTAC CTTATCCTCT TATATACT
[0109] 2041 ATTAGAATC AGTTTGATAC ACTAGAATC ACTAGAATC ACTAGGCGGA
[0110] 2101 GTRACKGG CLOCKGROUND CONNECTIVITY AACAAAAAAT AATAAAAAGT
[0111] 2161 CAAATACATG GAGCCACGCA ATGTTGTGAT AAATTTTCCG ATATACCACA AAAATTTGGA
[0112] 2221 TGTAGACCTT TTCGGGGGGA AAGTCGTTAT AAAATATATATATATAGACCTT
[0113] 2281 AAAACCTATA ACAGAAAAT AAGTCCACAA TTTAAAGGAA AAAAGTATA ACCATACAAA
[0114] 2341 CONNECTION ATTCTAAATA TAGAAAAAAAAAAAAAAAAAAAAATAAG AAAAGAAATA
[0115] 2401 AAAAAAAAAA ATTATTGTCC TAGAGAAAA AAAAATGCCA ATGTTGGATA TGTCAAGAG
[0116] 2461 AAGGACATTA TGCTAATGAA TGTCCAAATA AAAATAAAAG TAAAAAAAA GTAAAAGTAA
[0117] 2521 ATTACKS ATTACKGROUND ATTACKATC INTERFACE REPEAT RETURN
[0118] 2581 AAAATGAAAG TTATATTGT TTAGAAACA CTTCAGAATC TTTATATTTT GAAAATGAGT
[0119] 2641 INTERVAL ATAGTACTTT TATTAAAATTC AAAATAGCCC AAAAAATAAT TCTAGCTTAT
[0120] 2701 CONTACT CONTACT ATGTTTTT CONTROL CONTACT AACTTTTTGG
[0121] 2761 AAAGATTAA AGAACCTAT AAAAATTACA ATAGHAGATA AAAGAATTTCT AGAAATAAT
[0122] 2821 AAAGTTGCTT SERIES HELP AGAAAAAAA AATTCTTAGT ACCTACAT
[0123] 2881 TATCAATTTA ATGCATTAGE ACCTATTATT ATTACKTAG ATTTTCTTAG ATTACK
[0124] 2941 CCATTTTGTC AATATTTAAC TTATCACT CTTAAATGTC CTCAACTCAG TAATCAAAAA
[0125] 3001 CHAPTER TTAAAATTCC TAAATATCAT TCTTTTAAC TAAAATAAT AAGACAAATA
[0126] 3061 TGTTCCATATA TAATAATTT REQUEST ATGTTATT AAAATATAGA AGAAAAAATTA
[0127] 3121 CHAPTERS TTTCSEQUENCES TCTTTSEQUENCES AATAAAAATA CTACHINGS SEQUENCES
[0128] 3181 ATAAAGCTTA AAGACCCCTT AATAGCTTA AATAGTACCTA ATAATATCCC TTATTHEAD
[0129] 3241 NATIONATCA TGROUP AAAAGATG NATION GROUP TAGAAAAAG NATIONGROUP
[0130] 3301 CCTAGTAATA GTCCTCACTC ATATCATGC TGAAAAAA
[0131] 3361 CLOCKAAAA FREQUENCY SIDE AAAAAAAC CLOCK CLOCK
[0132] 3421 CCAAAAACAT THE NATIONAL ATAAAATATTAAAAGGGAAAAATGGTTT
[0133] 3481 TCCACCTTAG ATGTTAAATC AGCATATTGG CHAPTER TACATGAGA TACAAAACCA
[0134] 3541 TTAACAGCTT TTACTTTTCC ACCACAACAA CATTATGAAT GGAATTCATT ACCATTAGGT
[0135] 3601 TTAAACAAG CACCAGGAAT ATTTCAGAAT TTTATGAATA AACAATTACA AGGATTAGAA
[0136] 3661 AATATATGTA CAATATATAT AGATGATATA ATTATATTTT CTGAAGAAAA TAAAATTAAA
[0137] 3721 CACCTTAAAG ATGTTTTAAA AATAATAAAT AGATGTCAAG AAAAGGAAT TATTTTATCC
[0138] 3781 CAGCCAAAAG CTATTATTGC AAAAGAAAAA ATAGATTTTT TAGGATTAAA TATTTCTTCA
[0139] 3841 GGAGAAATTA TTTTACAAAA ACATATTGTT GAAAAAATTAA ATTTATTTCC TGATAAAATA
[0140] 3901 GAAAACAGAA AACAATTACA AAGATTTTTA GGAAATCTTA ATTACATAAG TGATCAAGGT
[0141] 3961 TTCTTAAAAA ATCTAGCAAA AGCTAGACGT CCCTTACAGA AAAAATTATC AGAAAAAGTC
[0142] 4021 CCTTGGACTT GGACTGATAA AGACAGCAGA TATGTTCAGG AGTTAAAAGC ATCTTGTATA
[0143] 4081 AATCTACCCA GATTATACAA TGCTAATATT CAGGATTTAT TAATAGTTTC TACTGATGCC
[0144] 4141 AGCAATGGTC ATTGGGGAGC TATTATGACA GCAATACCTC AAGAAATATT GCAAGAATAC
[0145] 4201 AAAGACAAA ATTACK TATTCTAAAT GAAAATCATT ATTACK GCAGTTATTC
[0146] 4261 AATACAAAAA CCTTTCAAAA CCTAGAAAAA TTACCAAAT ATACCAGTGG TACCTTTACT
[0147] 4321 GATACAGAAA CHAPTERACCC TATACACCAT CTAGAACCC TTGCAGCAAT TCGAACCTTC
[0148] 4381 AGAAAATGGA AAATTGATTT ACTACCAAAA CCTTTTATTT TAAAACCGA TTCCAAATAC
[0149] 4441 CTATCTGGAT TCTTAAAATA TAACATCAAA GCCAACTACA ATCAAGGACG ATTAATTAGA
[0150] 4501 TGGCAGCTAG AATTAAATCA GTATGATTAC AAGGTATT TAATTAAGAG TACAGATAAT
[0151] 4561 TTTGCAGCAG ATACTTTGAC TCGAGAATGG AAAGAGCAAT AAAGACATTG GAAAATCAAA
[0152] 4621 TTCAGCAAAA AGAAAATCAG ATCCAGCAAA AAATGAAGAA ATTAGAAG TTGCAAAAATG
[0153] 4681 AATTAAAAT TTTGAAAGAA ACATTTTCAG TGCTCAGAGG AAATCCTATA AAAGAAATTA
[0154] 4741 CONNECTED AATTCCHANGE CONNECTEDGATC AAATAAGTAT CONNECTED INTERRUPTED
[0155] 4801 AGAAGAAAGC TTTTTATGTT ATTACKAGG QUESTIONGAG AGGAATTTAT AGAINGGC
[0156] 4861 ATAAAACAGC ATTCCATA CHAPTER CHAPTERTCCA CHAPTER REQUIREMENT
[0157] 4921 AAGAAGCCCA GCAAGCTTTA AAAAATAGTC AATTCAGCAA TATTGTCCAA CAAAAAAC
[0158] 4981 CHANGE ATGTTTTGGA GAYCTCAGG CHANGE GAATAAGGC TATAATACTG
[0159] 5041 CCAGAAGCCT CTATAAATAT GCATCAAACT CTCCTGTTTG AGGGGAGCGA AAATTTAAAT
[0160] 5101 AAGAAAACCT TTGTGTTTTA TAAACTCTTT CGAGTTTTTT CAGATCTTTT GATCTATTTC
[0161] 5161 GAGTTAGTCG TGAGGTAATA CTCTGGACGA CACCAACA TAATTTCATC AACAGAACCC
[0162] 5221 AAAAACAGGT ATTTATTT ATAAAATAAT GTCAAAATTT TCTGCGTTTT HELP
[0163] 5281 CONTAINING TACTTAGCAA GCATGATATA ATTATGTCCA ATAAAGCATG
[0164] 5341 TATTATTAT ATGATTAGE GTTTAGEAAG ASSISTANCE TATTATTATTATTAGATCA TTACTAGA
[0165] 5401 TCGTATTGAA TTATACGATT TTAGTATAAA AACAGGTACG AGCACGTTCA ACTGTGTTTT
[0166] 5461 CAAACACGGT GTGCAACTAC AATGGAAAAT ACTAGAGAAA ACAATCAAGA AATAAAAGAA
[0167] 5521 TATAATATTG AAGAATTTTT CGATATATA CAAAATAAAG AAACTAGTTC TTATTATGGT
[0168] 5581 TTAGATATTC AAGTATAAG ATTAAATATA AAAAGTATAG AAAAACAAGG AAAAATTACA
[0169] 5641 TTAAAAGATG GTCATAGE TAAAATCCCT AAATTACTAA TGTTTAGAAA AAATATTA
[0170] 5701 TACTTAGGA ATTCCATTGT AGACAACCC ATAGAATAA ATACTTCACA AGGGCAATA
[0171] 5761 GCCTTATCAC TTATCAATCA ACAACAAATT TTATCAC TTGATAAAAT AAAAGTAGA
[0172] 5821 CONTINUOUS CONTINUOUS TARINE CONTINUOUS TAXATTC AAATTACT TAAAAGCACC
[0173] 5881 TATTCCGCTA ATTACK CTCC AATAGATTTA GCATATAG ACATTAGE ATTACTAA
[0174] 5941 AATCCTAAG AAAGAATTTTT NAMES AAAGAATC TTAAATCC AGTTAAAAA
[0175] 6001 TTATATA GTTSACK TGCCATTCCA TTATACA AAAATTTAGA TCAGTCATT
[0176] 6061 GGROUND ATAAATTTCA PROCESS CTTATGAAAA CHANGEGROUP TCCACTCAG
[0177] 6121 ATTACKTTATG STAGE TGCCCTAGT AATAGTCATC STAGE TTATAAAAAAC
[0178] 6181 AAAGATATA TCGAAATAGA ASSISTANT AAACAAACAAATACTTAGA ATTITUDES
[0179] 6241 VALLEY WINDOW TTTAATATTC HELP TTTAWINDOW
[0180] 6301 RECOMMENDATION RECOMMENDATION TCTACT COMMUNICATIONS CHARACTERISTICS
[0181] 6361 HELPCALL AAAAAAAA REFERENCE AATAAAAAAAAAAAAAAAAAAAAATAG
[0182] 6421 NATIONAL AAAAAA ACCATGCCA NATIONAL AAACGAATTT AAAATTAGE ATCCTTACA
[0183] 6481 GGAGAGTTTG ATTACKTTTT ATTACKGG AACTCGGACT TCAAAACATA TATTACK
[0184] 6541 ACTCAATAAC GGACCTGACT TTACATTTCA TTATAGTTTA TTAAAAGATA AAGACGGAAC
[0185] 6601 GGACTTGAAG TTACAATTAA ATGTAACAGA CTCGAAAACG ACATAAAGAA AAAGTTTGAA
[0186] 6661 AATGACAAAA A
[0187] NotV1 polymerase gene(SEQ ID NO:2)1965bp
[0188] 1 ATGAGTCAGA ATCAGAATAG TACTTTTATT AAAATCAAAA TAGCCCAAAA AATAATTCTA
[0189] 61 GCTTATATAG ATACAGGAGA TTCTTTATGT TTATTACAAG AAAATAATTT ACCAAAAACT
[0190] 121 TTTTGGAAAG AATTAAAGAA ACCTATAAAA ATAACAATAG CAGATAAAAG AATTCTAGAA
[0191] 181 ATAAATAAAG TTGCTTTATT AATAACCATA GTAATAAGAA ATAAAAAATT CTTAGTACCT
[0192] 241 ACAATATATC AATTTAATGC AGGAGTACCT ATGATTATAG GAAATAATTT TCTTAGATTA
[0193] 301 TATTATCCAT TTTGTCAATA TTTAACTTAT ATCACTCTTA AATGTCCTCA ACTCAGTAAT
[0194] 361 CAAAAACAAG AAATAATTAA AATTCCGATA TATCATTCTT TTAAACTAAA AATAATAAGA
[0195] 421 CAAATATGTT CCATAATAAA TAATTTAGAA GAAGAAATGT TAATTAAAAAA TATAGAAGAA
[0196] 481 AAATTACAAG AAAGATTTTC TAATGATCTT TTAGGAAATA AAAATACTAA TGAAGAATTA
[0197] 541 ATAGAAATAA AGCTTAAAGA CCCCTTACAA GAAGTAAATG TACCTAATAA TATCCCTTAT
[0198] 601 TCAGAAAAGG ATATCATTGA ATTTAAAAAA GATATGGATG ATTTAATAGA AAAAAGAGTT
[0199] 661 ATTAGACCTA GTAATAGTCC TCACTCAGCA CCAGCATTTT ATGTTAATAA TCATGCTGAA
[0200] 721 GAAAAAAGAG GAAAAAGAAG AATAGTTATT AATTATAAAA AAATGAATGA AGCTACAATA
[0201] 781 GGAACTCCAA AAACATTACC AAGAAAAGAT TATATAATAA ATAAATTAAA AGGGAAAAAA
[0202] 841 TGGTTTTCCA CCTTAGATGT TAAATCAGCA TATTGGCAAC TAAGATTACA TGAGAATACA
[0203] 901 AAACCATTAA CAGCTTTTAC TTTTCCACCA CAACAACATT ATGAATGGAA TTCATTACCA
[0204] 961 TTAGGTTTAA AACAAGCACC AGGAATATTT CAGAATTTTA TGAATAAACA ATTACAAGGA
[0205] 1021 TTAGAAATATATATATATATATATATATATATTCTGA AGAAAAATAAA
[0206] 1081 ATTACKACC TTAAAGATGT TTAAAATA ATTAAATAGAT GTCAAAAA NATION GATTATT
[0207] 1141 TTATCCCAGC CAAAAGCTAT TATTGCAAAA GAAAAAATAG ATTTTTAGG ATTAAATATT
[0208] 1201 TCTTCAGGAG AATTATTTT ACAAAAACAT ATTGTTGAAA AATTAAATTT ATTTCCTGAT
[0209] 1261 AAAATAGAAA HAZARD ATTACK TTTTAGAAA ATCTTAGAA HELP
[0210] 1321 CAAGGTTTCT TAAAAAATCT AGCAAAAGCT AGACGTCCCT TACAGAAAAA ATTATCGAA
[0211] 1381 AAAGTCCCTT GGACTTGGAC TGATAAGAC AGCAGATATG TTCAGGAGTT AAAAGCATCT
[0212] 1441 TGTATAAATC TACCCAGATT ATACAATGCT AATATTCAGG ATTTATTAAT AGTTTCTACT
[0213] 1501 GATGCCAGCA ATGGTCATTG GGGAGCTATT ATGACAGCAA TACCTCAAGA AATATTGCAA
[0214] 1561 CONNECTION ACAAAAATAA COMMUNITY CTAAATCAAA ATCATTCAAA COMMUNITYGAGAG
[0215] 1621 TTATTCAATA CAAAAACCTT TCAAAACCTA GAAAAATTAA CCAAATATAC CAGTGGTACC
[0216] 1681 TTTACTGATA CAGAAACAAG ATACCCTATA CACCATCTAG AAACCCTTGC AGCAATTCGA
[0217] 1741 ACCTTCAGAA AATGGAAAAT TGATTTACTA CCAAAACCTT TTATTTTAAA AACCGATTCC
[0218] 1801 AAATACCTAT CTGGATTCTT AAAATATAAC ATCAAAGCCA ACTACAATCA AGGACGATTA
[0219] 1861 ATTAGATGGC AGCTAGAATT AAATCAGTAT GATTACAAGG TATTATTAAT TAAGAGTACA
[0220] 1921 GATAATTTTG CAGCAGATAC TTTGACTCGA GAATGGAAAG AGCAATAA
[0221] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.
Claims
1. A Soymovirus, characterized in that: The genome of the virus is a covalently closed circular double-stranded DNA, and the virus contains the nucleotide shown in SEQ ID NO: 1 or a nucleotide that is completely complementary to the nucleotide shown in SEQ ID NO:
1.
2. An isolated nucleotide, characterized in that, Selected from at least one of the following nucleotides: The nucleotide represented by SEQ ID NO: 1; Nucleotides that are completely complementary to the nucleotide shown in SEQ ID NO:
1.
3. A primer pair, characterized in that, The primer pair is selected from any of the following primer pairs: The primer pairs shown in SEQ ID NO: 17 and 18; The primer pairs shown in SEQ ID NO: 19 and 20; The primer pairs shown in SEQ ID NO: 21 and 22; The primer pairs shown in SEQ ID NO: 23 and 24; The primer pairs shown in SEQ ID NO: 25 and 26.
4. The use of the primer pair according to claim 3 in the diagnosis of plant virus infection, characterized in that, The plant is a species of the genus *Pseudorasporium*; the species of the genus *Pseudorasporium* include any one or a combination of *Mabirium macranthum*, *Pseudorasporium thunbergii*, *Pseudorasporium odoratum*, *Pseudorasporium scaber ... and *Pseudorasporium pubescens*.
5. The use of the primer pair according to claim 3 in the identification of pathogens, characterized in that, The identification refers to: determining whether the pathogen to be tested is a Soymovirus as described in claim 1; or determining whether a Soymovirus as described in claim 1 exists in the sample.
6. The use of the primer pair according to claim 3 in the preparation of a pathogen detection device, characterized in that, The pathogen is a Soymovirus as described in claim 1; the detection device is in the form of a reagent or a chip.
7. A detection device, characterized in that, It comprises the primer pair as described in claim 3; the detection device is in the form of a reagent or a chip.
8. A method for detecting pathogens, comprising the steps of: 1) Provide the sample to be tested; the sample is derived from plants of the Cornaceae family; 2) Obtain the nucleotides of the sample to be tested; the nucleotides are DNA; 3) Contact the nucleotides of the sample to be tested or their amplification products with the primer pair as described in claim 3; 4) Determine whether the isolated nucleotides as described in claim 2 exist.