WO2018197733A1 - Procédé et nécessaire destinés à détecter un virus appartenant au genre potyvirus - Google Patents

Procédé et nécessaire destinés à détecter un virus appartenant au genre potyvirus Download PDF

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WO2018197733A1
WO2018197733A1 PCT/ES2017/070259 ES2017070259W WO2018197733A1 WO 2018197733 A1 WO2018197733 A1 WO 2018197733A1 ES 2017070259 W ES2017070259 W ES 2017070259W WO 2018197733 A1 WO2018197733 A1 WO 2018197733A1
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virus
mosaic
seq
mosaic virus
genus
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PCT/ES2017/070259
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Jesús Ángel SÁNCHEZ NAVARRO
Vicente PALLÁS BENET
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Consejo Superior De Investigaciones Científicas (Csic)
Universitat Politècnica De València
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/34011—Potyviridae
    • C12N2770/34022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to probes, particularly gender probes, methods and assays that are used for the simultaneous detection and / or quantification, in a single step, of a plurality of virus nucleic acid sequences belonging to the genus Poyvirus. More particularly, the invention relates to probes and methods for detecting and / or quantifying, by non-radioactive molecular hybridization, a plurality of virus nucleic acid sequences belonging to the genus Poyvirus, and also new virus subtypes of this genus. Therefore, the present invention relates, in general, to the field of virus detection.
  • Po ⁇ yvirus is a genus of viruses in the family Po ⁇ yviridae and represents one of the most economically important and widely distributed groups of plant viruses. Members of this genus can produce significant losses in agricultural, horticultural and ornamental crops.
  • the type member of the genus Po ⁇ yvirus, potato virus Y (PVY), together with the potato virus A (PVA; genus Poyyvirus) and the potato leaf curl virus (genus Poierovirus), represents the greatest threat for the production of potatoes throughout the world and can reduce crop yields up to 90%.
  • the sharka virus (PPV; genus Po ⁇ yvirus) also has great economic importance as it produces the most destructive diseases of stone fruits worldwide.
  • ELISA enzyme-linked immunoabsorbent assays
  • Journal Viro ⁇ ogica ⁇ Methods, 161: 289-296) developed a real-time multiple RT-PCR, in a single tube, for the simultaneous detection of PLRV, PVX, PVS and the tanning virus of tomato (TSWV) in potato and tomato.
  • Agindotan e ⁇ ai. (Agindotan e ⁇ al, 2006. Journal Viro ⁇ ogica ⁇ Methods, 142: 1-9) developed a method of multiple real-time RT-PCR using TaqMan® chemistry for the simultaneous detection of four potato viruses: PLRV, PVA, PVX and PVY.
  • these real-time multiple RT-PCRs do not include any internal control or reference gene.
  • there are technical difficulties for multiple PCR such as loss of sensitivity to increase the number of initiators in the reaction mixture or the need to adjust the position of the primers in the gene to be amplified in order to obtain easily distinguishable fragments. by size when analyzed by agarose gel electrophoresis.
  • several degenerate primers have been designed to recognize conserved regions of viral genomes of many virus species or the genus or entire family of viruses. (Rose et ai, Nucleic Acid Research. 1998; 26: 1628-1635).
  • viral genomes can be detected by hybridization methods using probes or oligonucleotides with a sequence homologous to the virus possibly present in the sample to be analyzed. These techniques are usually simple and non-radioactive as disclosed, for example, by fluorescence or chemiluminescence.
  • the methods based on hybridization techniques in addition to the advantages of specificity and sensitivity together with the possibility of being able to quantify the amount of viral nucleic acids present in the sample (viral load), allow the detection of different pathogens. In this way, this technology allows the multiple detection of plant viruses by means of a cocktail of specific probes (Sánchez-Navarro JA, et al., Journal of Virological Methods.
  • the invention provides a solution to the constant need for probes and methods to detect, identify and / or quantify simultaneously, quickly, specifically and sensitively, the complete series of viruses belonging to the genus Potyvirus that are present in a single sample.
  • the invention is based on the development of a hybridization system, preferably a non-radioactive hybridization system, in which different specific nucleotide sequences to be tested have been immobilized on a solid support, or diluted in a hybridization solution. and tested with gender specific probes (gender probes).
  • genus probes are designed to specifically hybridize with target sequences present in the Potyvirus genome, these target sequences being highly conserved regions of viral genomes of the members that belong to a virus genus, in the present invention preferably the genus Potyvirus, which allows the simultaneous detection of the complete series of viruses of this genus, preferably the genus Potyvirus, in a single test or test.
  • This system can be adapted to the development of user-friendly kits.
  • the invention provides a solution for the detection of a large number of viruses using gender probes with a reduced size, avoiding the problem associated with the use of large poiisondas in which each pathogen must be represented with the corresponding nucleotide sequence.
  • the gender probes of the present invention comprise, arranged in tandem, several nucleotide sequences, preferably at least three, more preferably at least five and even more preferably at least seven, from highly conserved regions of viruses belonging to the genus Potyvirus . It is known that there are 148 species described in the databases within the genus Potyvirus. The inventors have selected seven highly conserved regions distributed by the phylogenetic tree of this genus, specifically the seven highly conserved regions are located in the nuclear inclusion gene b (Nlb) of lettuce mosaic virus (LMV), mosaic virus watermelon (WMV), potato virus Y (PVY), pepper vein mottle virus (PVMV), sharka virus (PPV), soft potato mottle virus (SPFMV) and engraving virus tobacco (TEV).
  • LMV lettuce mosaic virus
  • WMV mosaic virus watermelon
  • PVY potato virus Y
  • PVMVMV pepper vein mottle virus
  • SPFMV soft potato mottle virus
  • TMV engraving virus tobacco
  • the highly conserved regions of the Nlb gene mentioned above comprise approximately 500 nucleotides each. Particularly preferred is the highly conserved region of the Nlb gene of the LMV comprising SEQ ID NQ: 7, of the WMV comprising SEQ ID NO: 6, of the PVY comprising SEQ ID NO: 5, of the PVMV comprising SEQ ID NO: 4, of! PPV comprising SEQ ID NO: 3, of the SPFMV comprising SEQ ID NO: 2 and of the TEV comprising SEQ ID NO: 1 (Fig. 1).
  • the term "highly conserved region,” as used herein, refers to a consensus gene section or relatively invariable or "conserved" amino acid sequence compared to other sections of the sequence between several species of the same genus or family. .
  • the highly conserved region for example, the CP region and the Nlb region, as shown in Fig. 1A, are highly conserved regions in the Potyvirus genome.
  • Potyvirus is the largest genus of plant viruses that produce significant losses in a wide range of crops.
  • the genus Potyvirus comprises 148 species of viruses of which some are economically important. Potyviruses are transmitted by aphids in a non-persistent manner and some of them are also transmitted by seeds. This genus includes many relevant species such as PPV, PVY, SPFMV, virus! dwarfing corn mosaic (MDMV), sugarcane mosaic virus (SCMV) or turnip mosaic virus (TuMV).
  • Fig. 2 shows that there is a clear hybridization signal in the samples when the temperature is 80 ° C, but more preferably 55 ° C, and also when the samples have at least 68% sequence identity compared to single or individual gender probes, without showing hybridization signal in healthy plant samples.
  • the inventors show that cross hybridization was also observed with the GP3, GP5 and GP7 genus probes of the present invention (Fsg.
  • the quantities detectable with the probes of genus GP3 (SEQ ID NO: 8), GP5 (SEQ ID NO: 9) and GP7 (SEQ ID NO: 10) of the invention are in the range of picograms, which is comparable to those obtained with single or individual gender probes comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, each individually.
  • the gender probes of the present invention which comprise the minimum number of highly conserved regions of the Nlb gene of the Potyvirus genus, are useful for detecting and / or quantifying the complete series of viral species included in the Potyvirus genus, and also new virus subtypes of this genus, if these species have at least 88% sequence identity with respect to the gender probes of the present invention. Therefore, the use of at least one of the genus probes of the invention, preferably the use of GP3, GP5, GP7 or any combination thereof, is useful for detecting all species of the Potyvirus genus.
  • the present invention refers to an isolated polynucleotide sequence (hereinafter referred to as an isolated probe or isolated gender probe of the invention) comprising nucleotide sequences selected from: SEQ ID NO: 1 , SEQ ID NO: 2 SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the present invention relates to an isolated polynucleotide sequence (referred to in the following or polynucleotide of the invention or genus probe of the invention) comprising, arranged in tandem, nucleotide sequences selected from: SEQ ID NO: 1, SEQ ⁇ D NO: 2 and SEQ ID NO: 3.
  • the polynucleotide sequence comprises SEQ ID NO: 8.
  • the polynucleotide sequence consists of SEQ ID NO : 8.
  • nucleotide sequence of the invention refers to polymeric forms of nucleotides of any length, either ribonucieotides or deoxyribonucleotides.
  • SEQ ID NO: 1 comprises the reverse and complementary sequence of nucleotides located between positions 7468 and 7947 of the TEV with NCBI number DQ986288;
  • SEQ ID NO: 2 comprises the reverse and complementary sequence of nucleotides located between positions 8563 and 9056 of the SPFMV with NCBI number KU511268;
  • SEQ ID NO: 3 comprises the reverse and complementary sequence of nucleotides located between positions 7495 and 7993 of the PPV with NCBI number U508427;
  • SEQ ID NO: 4 comprises the reverse and complementary sequence of nucleotides located between positions 7530 and 8009 of the PVMV with NCBI number DQ645484;
  • SEQ ID NO: 5 comprises the reverse and complementary sequence of the nucleotides located between positions 7510 and 7982 of the PVY with NCBI number AJ890346;
  • SEQ ID NO: 6 comprises the reverse and complementary sequence of the nucleotides located between positions 7882 and 8348 of the WMV with NCBI number
  • the polynucleotide sequence of the invention further comprises, also arranged in tandem, at least two, at least three or at least four of the nucleotide sequences selected from the list consisting of: SEQ ID NO: 4 , 5, 6, 7 and / or any combination thereof.
  • the polynucieotide further comprises SEQ ⁇ D NO: 4 and 5, or SEQ ⁇ D NO: 4, 5, 6 and 7.
  • the polynucieotide of the invention comprises SEQ ID NO : 9 or SEQ ID NO: 0,
  • the polynucieotide consists of SEQ ID NO: 9 or SEQ ID NO: 10.
  • tandem refers to the existence in the same DNA or protein sequence of at least three or more different fragments (nucleotides or amino acids) close to each other.
  • tandem refers to at least three fragments selected from SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7, preferably at least three fragments comprising the SEQ ID NO: 1, 2 and 3, resulting in the nucleotide sequence comprising SEQ ID NO: 8.
  • tandem refers to at least five fragments that they comprise SEQ ID NO: 1, 2, 3, 4 and 5, resulting in the nucleotide sequence comprising SEQ ID NO: 9, and, in a further embodiment, the term “in tandem” refers to at least seven fragments comprising SEQ ID NO: 1, 2, 3, 4, 5, 8 and 7, resulting in the nucleotide sequence comprising SEQ ID NO: 10.
  • the polynucieotidic sequence of the present invention preferably SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, is a probe, more particularly it is a gender probe.
  • the term “probe” refers to a nucleotide sequence that binds through a base pairing complementary to a sub-sequence of a target nucleic acid.
  • the term “gender probes” refers to a probe that carries different conserved regions of a specified viral genus that are fused in tandem and has the ability to hybridize with all members of the genus.
  • the gender probes of the present invention hybridize, at a temperature of a range of at least 50 ° C to 100 ° C, optionally in the range of at least 55 ° C to 90 ° C, 55 ° C to 85 ° C, 55 ° C to 80 ° C, 55 ° C to 75, 55 ° C to 70, 55 ° C to 70, 55 ° C to 68 ° C, 55 ° C to 65 ° C, 55 ° C to 60; with all members of the gender comprising at least 68% sequence identity with respect to the gender probes of the present invention.
  • the temperature for hybridization with all members of the genus Potyv ⁇ rus is at least 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 67 ° C, 68 ° C, 69 ° C , 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C or 75 ° C. More preferably, at least 50 ° C and more preferably 50 ° C.
  • sequence identity is at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. More preferably, the sequence identity is at least 68% with respect to the gender probes of the present invention.
  • the present invention relates to the in vitro use of the genus or polynucieotide probes of the present invention, to detect and / or quantify viruses belonging to the genus Potyvirus, preferably in a single stage and, more preferably, in a single isolated sample.
  • the in vitro use of the gender probes of the present invention is carried out at the above-mentioned hybridization temperature, preferably at least 50 ° C.
  • sample refers to a subject! gene amplified from the tai subject as an amplified total nucleic acid of a test subject in which a species of target virus is detected or identified.
  • total RNA ribonucleic acid
  • the sample is preferably a plant or plant sample.
  • plant sample or “plant sample” refers to any processed or unprocessed material from, in whole or in part, a plant.
  • a plant material can be a part of a plant, a seed, a fruit, a leaf, a root, a plant tissue, a crop of plant tissue, a plant explant, a plant cell or a plant whole
  • a sample of a plant material may refer to a fraction or part of the plant material, for example, a fraction or part to be analyzed by a method according to the invention to extrapolate a result to the matter! of the total plant from which the sample was obtained.
  • a sample of a plant material can also refer to the material itself! of plant, if all plant material is subjected to analysis.
  • the present invention relates to a method for the detection and / or quantification in vitro of the complete series of viruses belonging to! Potyvirus genus in an isolated sample of an infected subject, comprising the stages of:
  • the test sample in step a), can be homogenized with a buffer.
  • the buffer comprises 50 mM sodium citrate (pH 8.5 ⁇ and 5 mM EDTA.
  • step b) comprises hybridization with dot blotting.
  • the test sample and the gender probe of the invention are preferably immobilized on a nylon membrane, preferably on a positively charged nylon membrane.
  • the immobilization is preferably carried out by temperature or by UV light.
  • the mixture comprising the test sample and the genus probes of the invention is incubated at a temperature range of at least 50 ° C to 100 ° C, optionally in the range of at least 55 ° C to 90 ° C, 55 ° C to 85 ° C, 55 ° C to 80 ° C, 55 ° C to 75, 55 ° C to 70, 55 ° C to 70, 55 ° C at 68 ° C, 55 ° C to 85 ° C or 55 ° C to 60 ° C.
  • the temperature for hybridization with all Potyvirus genus members is at least 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 67 ° C, 68 ° C, 69 ° C , 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C or 75 ° C. More preferably, at least 50 ° C and more preferably 50 ° C.
  • hybridization between the genus probes of the invention and the test sample indicates that the test sample comprises a Potyvirus.
  • hybridization between the genus probes of the invention and the test sample indicates that the Potyvirus comprises a sequence identity of at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with respect to the gender probes of the present invention. More preferably, the sequence identity is at least 68% with respect to the gender probes of the present invention.
  • Potyviruses are selected from the list consisting of: Agropyron mosaic virus, Algerian watermelon mosaic virus, celery virus Y, arracacha mottle virus, banana bract mosaic virus, Baseila rugosa mosaic virus, common bean necrotic mosaic virus, common mosaic virus the bean, yellow bean mosaic virus, beet mosaic virus, Bidens mosaic virus, Bidens mottle virus, Chamaesciila corymbosa virus A, Brugmansia suaveolens mottle virus, yellow achira striatum virus , celery mosaic virus, chili ring spot virus, chili vein mottling virus, yellow clover vein virus, dactyl striatum virus, colombian datura virus, cowpea mosaic virus transmitted by aphids, Daphne mosaic virus, malanga mosaic virus, East Asian Passifiora virus, Freesia mosaic virus, Liar ⁇ a Friti virus, Habenaria mosaic virus, mosaic
  • the present invention refers to a kit or device for detecting and / or quantifying viruses belonging to the genus Potyv ⁇ rus comprising the poiinucieotide or gender probes of the present invention, in an isolated sample.
  • kits or device may contain all the reagents necessary to detect and / or quantify the virus belonging to the genus Potyv ⁇ rus, preferably by any of the methods known in the state of the art and / or by the method disclosed herein.
  • the kit may also include, without imitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the necessary means and containers for the implementation and optimization.
  • the kit further comprises instructions for performing any of the methods of the invention.
  • microarray of the invention comprising the polynucleotides of the present invention, preferably comprising at least one of the nucleotides selected from the list consisting of: SEQ ID NO: 8, 9, 10 or any combination thereof; more preferably comprising SEQ ID NO: 8, SEQ ID NO: 9 and / or SEQ ID NO: 10.
  • the present invention relates to the use of the kit, or the microarray of the present invention, for detecting and / or quantifying viruses belonging to the genus Potyv ⁇ rus in an isolated sample.
  • kits or microarray can be used and the use is not particularly limited, although use in the method of the invention is preferred in any of its embodiments. Unless defined otherwise, all technical and scientific terms used in this document have e! meaning commonly understood by a person skilled in the art to which this invention belongs. In the practice of the present invention methods and materials similar or equivalent to those described herein may be used. Throughout the description and the claims, the word "comprises” and its variations should not be considered as extenders of other technical characteristics, additives, components or stages. Other objects, advantages and characteristics of the invention will be apparent to those skilled in the art upon examination of the description, or they can be learned through the implementation of the invention. The following examples, drawings and sequence listing are provided by way of illustration and should not be considered as imitators of the present invention. DESCRIPTION OF THE DRAWINGS
  • Fig. 1 Schematic representation of the gifts of GP3, GP5 and GP7 probes introduced into plasmid pSK.
  • the highly conserved region of the Nlb gene of each Potyv ⁇ rus (LMV, WMV, PVY, PVMV, PPV, SPF V and TEV) is indicated in each clone.
  • Fig. 2 Evaluation of cross hybridization between SEQ ID NO: 1 to 7.
  • The% identity refers to the percentage of identity estimated by the MatGAT program between SEQ ID NO: 3 and the rest of the Potyv ⁇ rus sequences mentioned above.
  • FIG. 3 Evaluation of cross hybridization between the seven simple Potyvirus probes comprising SEQ ID NO: 1 to 7 and the genus probes GP3 (SEQ ID NO: 8), GP5 (SEQ ID NO: 9) and GP7 (SEQ ID NO: 10).
  • A) Replicas of the membrane described in Figure 2A were hybridized with the GP3, GP5 and GP7 gender probe at 50 ° C.
  • 100 ng of total RNA extracted from melon (A), cucumber (B), N. benthamiana (C), tomato (D), chrysanthemum (E) and G. aurantiaca (F) were applied.
  • the numbers in the upper part of the left panel indicate the picograms of transcripts applied to the membrane.
  • the phylogenetic analysis was performed with 94 Potyviruses which have the complete polynucleotide sequence in the database (GenBank).
  • Phylogenetic analyzes were deduced in a multi-stage process: in the first stage, the sequences were aligned using the CLUSTAL W program (Higgins D., ei a /., Nuciese Acids Res. 1994, 22: 4673-4880) until , in a second stage, ios neighbor tree trees are generated using the JTT model, implemented in EGA7: Molecular Evolutionary Genetics Analysis version 7.0 for more data series large (Kumar S., et ai, Molecular Bio! ogy and Evo! ution. 2018, 33: 1870-1874).
  • Pepper yellow mosaic virus RNA strain NC_014327.1 66 DF
  • the plant tissue was obtained from the German Collection of Microorganisms and Cell Cultures DSMZ of the Leibniz Institute (https: / www.dsmz.de/home.html) or of the Mediterranean Agroforestry Institute of the Polytechnic University of Valencia (http: // www. upv.es/iam/ingles/bienvenida.html) (see Table 4).
  • Total nucleic acid extraction was performed using 0.1 g of leaf tissue using the silica capture extraction protocol (MacKenzie DJ, et al., Pla ⁇ . Disease. 1997, 81: 222-226) which purifies the acids total nuclei
  • the extracted nucleic acids were stored at -80 ° C until use.
  • the homogenized direct tissue was also tested with citrate buffer, the sample being extracted and the membrane applied as previously described (Sánchez-Navarro JA, et al., Plant Pathology, 1998, 47: 780-786; Sánchez -Navarro JA, et ai, Journal heard Viro ⁇ ogica ⁇ Methods. 1999, 82: 167-175.).
  • healthy and infected tissues were homogenized with 5 volumes of cold extraction buffer (50 mM sodium citrate, 5 mM EDTA, pH 8.5) and applied directly (1 ⁇ ) on nylon membranes.
  • Reverse transcription and PCR reactions were performed as previously described (Herranz M. C, et al, Journal o ⁇ Vim ⁇ ogicai Methods. 2005, 124: 49-55), using specific primers (Table 2), which contained the sites of Xhoi and Sai! 5 'and 3', respectively.
  • the PCR products were digested with the two restriction enzymes and extracted from the ge! of agarose.
  • the purified PCR fragments were inserted into the plasmid pBluescript SK (+), previously digested with the enzyme Xhoi and dephosphory.
  • GP3 SEQ ID NO: 8
  • GP5 SEQ ID NO: 9
  • GP7 SEQ ID NO: 10
  • GP3 contained three (PPV-SPF V-TEV), five (PVY-PV V-PPV- SPFMV-TEV) and seven (LMV-WMV-PVY-PVMV-PPV-SPFMV-TEV) pathogen sequences bound in tandem, respectively (Flg. 1).
  • CACACTCGAGGAAAGC 1 1 TEV DQ986288 TGGGAATTTGG 7468-7947
  • the numbers refer to! corresponding polynucleotide of the sequence available in the indicated access number of the GenBank database. Synthesis of gender probes labeled with digoxigenin and hybridization procedure
  • the seven DNA fragments cloned in plasmid pSK + were amplified by PCR using the corresponding antisense primer and reverse primer (Table 2 ).
  • the resulting PCR fragment contains the corresponding Potyvirus clone plus the T3 promoter.
  • the ampiicons were purified on agarose gei and then used directly for the synthesis of unlabeled transcripts complementary to the corresponding digoxigenin-labeled RNA probes.
  • Known quantities of the three free transcripts were serially diluted (five times) in sterile water and applied directly to nylon membranes. Hybridization with dot-blot transfer is performed as previously described.
  • Example 1 Evaluation of cross hybridization between single probes and gender probes of the present invention GP3, GP5 and GP7.
  • each individual or single Potyvirus genus probe (SEQ ID NOs: 1, 2, 3, 4, 5, 6 or 7) to cross-hybridize among themselves and also with the gender probes of the invention was analyzed.
  • complementary transcripts of the seven selected sequences were synthesized, SEQ ID NQ: 1 to SEQ ID NO: 7, which were serially diluted (1: 5) and applied on nylon membranes, to obtain a range of concentrations RNA between 200-0.06 pg / ⁇ .
  • RNA 100 nanograms extracted from melon (A), cucumber (B), Nicoiiana benthamiana (C), tomato (D), chrysanthemum (E) and Gynura aurantiaca (F) was applied to the membrane (Fig. 2A) ).
  • Replicas of the same membrane were hybridized first night with the PPV ribosonde (20 ng / mi in the hybridization solution) at different hybridization temperatures, to assess the effect of said parameter on cross hybridization (Fig, 2A). All the films were exposed between 15 and 30 minutes. No cross reaction was observed when hybridization was performed at 68 ° C.
  • sequences with an identity percentage below 65% with the corresponding probe were not detected at 55 ° C, but sequences with an identity percentage greater than 67% were detected, except the probe of PPV that It produced negative results with LIV1V, which shared 89.7% identity.
  • Different behaviors were observed between 85% and 87% of identity, suggesting that other factors influence cross-hybridization.
  • a correlation between the detection limit and the percentage of identity was also observed. In this way, sequences showing an identity percentage of 70% or higher could detect up to 1.9 pg by cross hybridization.
  • MO: 1 which led to SEQ ID NO: 8 could detect the seven Potyvirus sequences in which the lower detection limit corresponded to PVMV transcripts at 8 pg / ⁇ (Fig. 3).
  • the inclusion of two additional tandem viral sequences in the GP5 genus probes (SEQ ID NO: 5 (PVY), SEQ ID NO: 4 (PVMV), SEQ ID NO: 3 (PPV), SEQ ID NO: 2 (SPFMV ) and SEQ ID NO: 1 (TEV) leading to SEQ ID NO: 9) allowed the detection of the seven viral sequences with better detection limits, at least for the sequences present in the GP5 specific gender probes.
  • the membrane was hybridized with GP7 (SEQ ID NO: 7 (LV), SEQ ID NO: 6 (WMV), SEQ ID NO: 5 (PVY), SEQ ID NO: 4 (PVMV), SEQ ID NO: 3 (PPV), SEQ ID NO: 2 (SPFMV) and SEQ ID NO: 1 (TEV) leading to SEQ ID NO: 10) and the data obtained were the same as ios obtained with the same detection limit for all ios virus analyzed, corresponding to 0.32 pg / ⁇ .
  • GP5 the increase in the size of the gender probe affected the limit of detection, reducing five times the best signals obtained with GP3 or GP5.
  • 3B shows a summary of the detection limit obtained with the three gender probes in which the gray colors indicated the last number of transcripts showing hybridization with the corresponding GP probe.
  • the corresponding picograms (pg) of transcripts assigned for each gray color are indicated on the right side of Fig. 3B.
  • White indicates absence of hybridization signal.
  • Example 2 The GP3, GP5 and GP7 genus probes of the invention can detect all Potyvirus strains.
  • Fig. 2 and 3 indicate that sequences showing a percentage of identity of 68% or higher can be detected with the gender probes selected by cross hybridization.
  • a MatGat analysis was performed using the equivalent region of the 94 Potyvirus strains (Table 1) (Campanelia JJ, the ai. BMC Bioinformatics. 2003, 4: 29).
  • GP3 presents the most unfavorable situation for the detection of PVIV1V since its percentage of identity with the three cloned sequences present in GP3 is approximately 66-68% (Fig. 2 and Table 3).
  • the onion dwarf yellowing virus which theoretically could only be detected with the PPV clone (SEQ ID NO: 3 ⁇ (69.3% identity percentage), was detected with the three probes of gender.
  • Etching virus Physalis HH / HH + / + + / + of floridana tobacco (TEV)
  • +, - and * correspond to the positive, negative and doubtful results, respectively, nf, fresh tissue is not available.

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Abstract

La présente invention concerne, en général, des sondes de genre, des procédés et des nécessaires pour détecter et identifier, en une seule étape, tous les virus connus qui appartiennent au genre Potyvirus, ainsi que des nouveaux sous-types de virus de ce genre, au moyen de sondes qui reconnaissent des séquences de nucléotides de Potyvirus conservées par hybridation moléculaire non radioactive.
PCT/ES2017/070259 2017-04-27 2017-04-27 Procédé et nécessaire destinés à détecter un virus appartenant au genre potyvirus WO2018197733A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578627A1 (fr) * 1992-07-09 1994-01-12 Monsanto Company Plantes résistantes aux virus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0578627A1 (fr) * 1992-07-09 1994-01-12 Monsanto Company Plantes résistantes aux virus

Non-Patent Citations (29)

* Cited by examiner, † Cited by third party
Title
AGINDOTAN ET AL., JOURNAL VIROLOGICAL METHODS, vol. 142, 2006, pages 1 - 9
AGINDOTAN ET AL: "Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan<(>R) real-time RT-PCR", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 142, no. 1-2, 19 April 2007 (2007-04-19), pages 1 - 9, XP022034322, ISSN: 0166-0934, DOI: 10.1016/J.JVIROMET.2006.12.012 *
ANA PEIRÓ ET AL: "Simultaneous detection of eight viruses and two viroids affecting stone fruit trees by using a unique polyprobe", EUROPEAN JOURNAL OF PLANT PATHOLOGY, KLUWER ACADEMIC PUBLISHERS, DO, vol. 132, no. 4, 20 November 2011 (2011-11-20), pages 469 - 475, XP035022458, ISSN: 1573-8469, DOI: 10.1007/S10658-011-9893-0 *
CAMPANELLA J. J. ET AL., BMC BIOINFORMATICS., vol. 4, 2003, pages 29
CAMPANELLA J.J. ET AL., BMC BIOINFORMATICS, vol. 4, 2003, pages 29
HERRANZ M. C. ET AL., JOURNAL OF VIROLOGICAL METHODS., vol. 124, 2005, pages 49 - 55
HERRANZ M.C. ET AL., JOURNAL OF VIROLOGICAL METHODS, vol. 124, 2005, pages 49 - 55
HIGGINS D., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 4680
KUMAR S. ET AL., MOLECULAR BIOLOGY AND EVOLUTION, vol. 33, 2016, pages 1870 - 1874
MACKENZIE D. J. ET AL., PLANT DISEASE., vol. 81, 1997, pages 222 - 226
MACKENZIE D.J. ET AL., PLANT DISEASE., vol. 81, 1997, pages 222 - 226
MAS P. ET AL., JOURNAL OF VIROLOGICAL METHODS., vol. 45, 1993, pages 93 - 102
MORTIMER-JONES ET AL., JOURNAL VIROLOGICAL METHODS, vol. 161, 2009, pages 289 - 296
MORTIMER-JONES S M ET AL: "A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 161, no. 2, 1 November 2009 (2009-11-01), pages 289 - 296, XP026501545, ISSN: 0166-0934, [retrieved on 20090709], DOI: 10.1016/J.JVIROMET.2009.06.027 *
NIE; SINGH, JOURNAL VIROLOGICAL METHODS., vol. 86, 2000, pages 179 - 185
PALLÁS V. ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 81, 1998, pages 461 - 468
PEIRÓ A. ET AL., EUROPEAN JOURNAL OF PLANT PATHOLOGY., vol. 132, 2012, pages 469 - 475
PEIRÓ ET AL., EUROPEAN JOURNAL OF PLANT PATHOLOGY, vol. 132, 2012, pages 469 - 475
ROSE ET AL., NUCLEIC ACID RESEARCH, vol. 26, 1998, pages 1628 - 1635
SANCHEZ-NAVARRO ET AL., PLANT PATHOLOGY, vol. 47, 1998, pages 780 - 786
SÁNCHEZ-NAVARRO J. A. ET AL., JOURNAL OF VIROLOGICAL METHODS, vol. 82, 1999, pages 167 - 175
SÁNCHEZ-NAVARRO J. A. ET AL., PLANT PATHOLOGY, vol. 47, 1998, pages 780 - 786
SÁNCHEZ-NAVARRO J. A. ET AL., PLANT PATHOLOGY., vol. 47, 1998, pages 780 - 786
SANCHEZ-NAVARRO J.A. ET AL., JOURNAL OF VIROLOGICAL METHODS, vol. 82, 1999, pages 167 - 175
SANCHEZ-NAVARRO J.A. ET AL., JOURNAL OF VIROLOGICAL METHODS., vol. 82, 1999, pages 167 - 175
SCHOLTHOF ET AL., MOLECULAR PLANT PATHOLOGY, vol. 12, 2011, pages 938 - 954
WEI T ET AL: "Development of a short oligonucleotide microarray for the detection and identification of multiple potyviruses", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 162, no. 1-2, 1 December 2009 (2009-12-01), pages 109 - 118, XP026682327, ISSN: 0166-0934, [retrieved on 20090803], DOI: 10.1016/J.JVIROMET.2009.07.024 *
ZHIYOU DU ET AL., PLANT DISEASE., vol. 90, 2006, pages 185 - 189
ZHIYOU DU ET AL: "Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control", PLANT DISEASE, vol. 90, no. 2, 1 February 2006 (2006-02-01), pages 185 - 189, XP055055760, ISSN: 0191-2917, DOI: 10.1094/PD-90-0185 *

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