WO2018197733A1 - Methods and kit for the detection of viruses belonging to the potyvirus genus - Google Patents

Abstract

The present invention relates generally to genus probes, methods and kits for detecting and identifying, in a single step, all the known viruses belonging to the Potyvirus genus, and also new subtypes of viruses from this genus, using probes which recognize conserved Potyvirus nucleotide sequences by means of non-radioactive molecular hybridization.

Description

 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.

BACKGROUND TECHNIQUE Poíyvirus is a genus of viruses in the family Poíyviridae 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 Poíyvirus, 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 Poíyvirus) also has great economic importance as it produces the most destructive diseases of stone fruits worldwide. Given these damaging properties, the reason that PVY and PPV have been selected among the top ten plant viruses with maximum economic and scientific importance is clear (Scholthof eí al., Molecular Plañí Paíhology 2011, 12: 938-954) . The fact that the Poyviruses are spread by aphids, and that each Poyvirus can be transmitted by many different species of aphids and that each species of aphid can transmit many Poyyviruses, makes it even more difficult to fight and prevent infection by Poyyviruses in agriculture . Therefore, there is a need for methods and assays to identify infected cultures.

Most of the available systems use a combination of inspections of field of visual symptoms and serological tests using enzyme-linked immunoabsorbent assays (ELISA). However, these methods take time and are expensive. In addition, assays that detect viral genomes, in general, are based on hybridization methods and amplification methods. Genomic amplification methods, such as reverse transcription (RT) followed by polymerase chain reaction (PCR), allow certain viral genome sequences to be amplified several million times, so that they can be easily detected. Multiple RT-PCR has been introduced as a means to reduce cost and increase efficiency. For example, the detection by multiple RT-PCR of five potato viruses (PVA, PVS, PVX, PVY and PLRV) has also been reported (Nie and Singh, Journal Viroíogicaí Methods. 2000, 86: 179-185; Zhiyou Du , et al., Plant Disease. 2006, 90: 185-189). However, these methods are based on electrophoresis in amplicon agarose gei, which is not very suitable for high performance applications. Multiple real-time RT-PCR also reduces the cost of virus detection and facilitates high virus detection. Mortimer-Jones eí a !. (Mortimer-Jones et a !. 2009. Journal Viroíogicaí 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 eí ai. (Agindotan eí al, 2006. Journal Viroíogicaí 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. However, these real-time multiple RT-PCRs do not include any internal control or reference gene. In addition, the primers developed by Agindotan eí ai. which target PVY may not detect some viral strains of PVY, since variation of nucleotides has been observed at the primer hybridization sites after multiple genome alignments. In addition, 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. Accordingly, 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).

In addition, 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. 1999, 82: 167-175) or by a single probe that carries the different viral fragments fused in tandem or poiisonda (Herranz MC, et ai, Journal oí Virological Methods. 2005, 124: 49-55). The poiisondas allow the detection of several pathogens with a detection limit similar to that obtained for the individual probes (Peiró eí al., European Journal oí Plañí Paíhology. 2012, 132: 469-475). However, this approach requires the introduction of a specific probe for each pathogen to be detected, which increases the size of the corresponding probe when a large number of different pathogens have to be detected. This aspect, together with the necessary reduction of the hybridization temperature, restricts the number of different pathogens that could be detected.

Therefore, the need for tools to detect, identify and / or quantify simultaneously, in a single test, all nucleic acid sequences of viruses belonging to the genus Potyvirus, and also new virus subtypes of this genus, persists. , by a sensitive, fast and reliable method, using gender-specific probes, which allow, as previously mentioned, the simultaneous detection of multiple Potyviruses with a reduced cost. SUMMARY OF THE INVENTION

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. For this, 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). These 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. In addition, 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). 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. . In accordance with the present invention, 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).

As shown by the examples included in the present invention, there is a great influence of temperature in the cross-hybridization analysis between the genus, single or individual probes of the invention, and the samples tested. 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. In addition, the inventors show that cross hybridization was also observed with the GP3, GP5 and GP7 genus probes of the present invention (Fsg. 3), with the advantage that this hybridization could be performed at 50 ° C without hybridization signals in the healthy plants Furthermore, it is interesting that 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. These results suggest that 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. To confirm this, the inventors analyzed different Potyvirus species (n = 32) in isolated field samples and the results obtained show that all Potyvirus analyzed were detected with simple genus probes of the invention (Table 3), or which confirms that ios 148 viruses of the genus Potyvirus can be detected in a single assay, with probes of the genus GP3, GP5, GP7, or any combination thereof, of the present invention.

Therefore, in a first aspect, 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.

Furthermore, in a second aspect, 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. In a preferred embodiment, the polynucleotide sequence comprises SEQ ID NO: 8. In a more preferred embodiment, the polynucleotide sequence consists of SEQ ID NO : 8.

Here, the terms "nucleotide sequence of the invention", "polynucleotide of the invention" and "oligonucleotide of the invention" are used interchangeably. These expressions refer to polymeric forms of nucleotides of any length, either ribonucieotides or deoxyribonucleotides. In a further embodiment, 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 EU660589 and SEQ ID NO: 7 comprises the reverse and complementary sequence of nucleotides located between positions 7961 and 8457 of the LV with NCBI number X97704,

In a preferred embodiment, 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. In a more preferred embodiment, the polynucieotide further comprises SEQ¡D NO: 4 and 5, or SEQ¡D NO: 4, 5, 6 and 7. In a further embodiment, the polynucieotide of the invention comprises SEQ ID NO : 9 or SEQ ID NO: 0, In a more preferred embodiment, the polynucieotide consists of SEQ ID NO: 9 or SEQ ID NO: 10.

According to the invention, the term "in 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. Particularly, in the present invention, the term "in 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. In a further embodiment, the term "in 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.

In a more preferred embodiment, 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.

According to the invention, 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. Preferably, 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. In a preferred embodiment, the temperature for hybridization with all members of the genus Potyvírus 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. In another preferred embodiment, the 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.

In another aspect, 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. In another preferred embodiment, 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.

A "sample", as used herein, 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. For example, total RNA (ribonucleic acid), preferably from a plant, is a type of the test sample described in the invention. In a preferred embodiment, the sample is preferably a plant or plant sample. As used herein, the term "plant sample" or "plant sample" refers to any processed or unprocessed material from, in whole or in part, a plant. For example and without limitation, 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.

In another aspect, 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:

 a) obtain a total nucleic acid preparation of the infected subject;

 b) contacting and incubating the preparation of nucleic acid of the previous stage with the gender probes of the present invention,

 c) detecting a hybridization between the genus probes of the invention and the test sample, where hybridization indicates that the target virus is a Potyvirus.

In a preferred embodiment of the method of the present invention, in step a), alternatively, the test sample can be homogenized with a buffer. In other preferred embodiment, the buffer comprises 50 mM sodium citrate (pH 8.5} and 5 mM EDTA.

In another preferred embodiment of the method of the present invention, step b) comprises hybridization with dot blotting. In another preferred embodiment, the test sample and the gender probe of the invention are preferably immobilized on a nylon membrane, preferably on a positively charged nylon membrane. In another preferred embodiment, the immobilization is preferably carried out by temperature or by UV light.

In another preferred embodiment of! method of the present invention, 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. In another preferred embodiment, 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.

In another preferred embodiment of the method of the present invention, hybridization between the genus probes of the invention and the test sample indicates that the test sample comprises a Potyvirus. In another preferred embodiment, 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.

Another preferred embodiment of the method of the invention is characterized in that the 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, Liaría Friti virus, Habenaria mosaic virus, mosaic virus of Hardenbergia, Hippeastrum mosaic virus, Hordeum mosaic virus, Iranian Aleppo sorghum mosaic virus, Japanese yam mosaic virus, Aleppo sorghum mosaic virus, Keunjorong mosaic virus, Konjac mosaic virus , yellow leek striatum virus, lettuce mosaic virus, lilium mottle virus, lupine mosaic virus, dwarf corn mosaic virus, watermelon Moroccan mosaic virus, narcissus degeneration virus, virus of late yellowing of narcissus, yellow striated virus of narcissus, dwarfing yellowing virus of onion, Ornithogaíum mosaic virus, Panax virus Y, papaya leaf distortion mosaic virus, ring spot virus Papaya, hardening fruit ios passion fruit virus, seed-transmitted pea mosaic virus, peanut mottle virus, Pennisetum mosaic virus, virus of pepper mottling, severe pepper mosaic virus, pepper vein mottle virus, yellow pepper mosaic virus, Peruvian tomato mosaic virus, sharka disease virus, phytolacca mosaic virus, virus Potato A, potato virus V, potato virus Y, chive mosaic virus, yellow shallot striatum virus, sorghum mosaic virus, soy mosaic virus, cane mosaic virus of sugar, sunflower chlorotic mottle virus, sweet potato sweet spotted virus, sweet potato latent virus, sweet potato virus 2, sweet potato virus C, sweet potato G virus, Telosma mosaic virus, Thunberg fritillary, tobacco engraving virus, tobacco vein banded mosaic virus, tobacco vein mottling virus, tomato necrotic dwarf virus, turnip mosaic virus, Vallota speciosa virus, Y verbena virus, watermelon mosaic virus, virus! Mosaic of the wild potato, virus! wild tomato mosaic, wisteria vein mosaic virus, ia jicama mosaic virus, soft yam mosaic virus, yam mosaic virus, soft creek mosaic virus, zucchini tabby mosaic virus and virus of the yellow zucchini mosaic.

In another aspect, the present invention refers to a kit or device for detecting and / or quantifying viruses belonging to the genus Potyvírus comprising the poiinucieotide or gender probes of the present invention, in an isolated sample.

Said kit or device may contain all the reagents necessary to detect and / or quantify the virus belonging to the genus Potyvírus, 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. On the other hand, the kit can include all the necessary means and containers for the implementation and optimization. Preferably, the kit further comprises instructions for performing any of the methods of the invention.

Another aspect of the invention relates to a matrix or microarray, hereinafter 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.

In another aspect, 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 Potyvírus in an isolated sample.

In this way, the endogenous control can also be included within the kit or microarray. The kit 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 Potyvírus (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. A) Influence of temperature at cross hybridization between PPV probe (SEQ D NO: 3) and the rest of the indicated Potyvírus sequences: LMV (SEQ ID NO: 7), PPV (SEQ ID NO: 3), PVMV (SEQ ID NO: 4), PVY (SEQ ID NO: 5), SPFMV (SEQ ID NO: 2 ), TEV (SEQ ID NO: 1), WMV (SEQ ID NO: 6). A: melon; B: cucumber; C: Nicotiana benthamiana; D: tomato; AND; Chrysanthemum and F: Gynura aurantiaca. The numbers on the top of the left panel indicate the picograms of transcripts applied to the membrane. 1 = 200 pg; 2 = 40 pg, 3 = 8 pg, 4 = 1, 6 pg, 5 = 0.32 pg and 6 = 0.006 pg. The% identity refers to the percentage of identity estimated by the MatGAT program between SEQ ID NO: 3 and the rest of the Potyvírus sequences mentioned above. B) Summary of the results obtained in A. The numbers represent the percentage of identity between SEQ ID NO: 3 (simple PPV probe) and the rest of the Potyvírus simple probes. The different textures indicated the last number of transcripts showing hybridization with the PPV probe (SEQ ID NO: 3). The Corresponding picograms (pg) of transcript assigned for each texture are indicated in the right part of the table. White indicates the absence of hybridization signal. C) Schematic representation of the hybridization observed with the seven single Potyvirus probes of SEQ ID NO: 1 to 7 of the invention. The numbers represent the corresponding percentage of identity assigned by the MatGAT program, while each texture represents the last number of transcripts showing hybridization with the corresponding probe, as indicated in B. The films were exposed for 15 min.

 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. For the healthy control, 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 films were exposed for 15 min. B) Summary of the results obtained in A. The different textures indicate the last number of transcripts that show hybridization with the corresponding gender probe. The corresponding picograms (pg) of transcript assigned for each shade of gray are indicated on the right side of the table. White indicates absence of hybridization signal.

MATERIAL EXAMPLES AND METHODS

IT analysis

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). Third, the statistical reliability of trees constructed by the bootstrap method based on 10,000 pseudoreplications was evaluated. The most conserved region observed in the Potyvirus alignment, which covers 500 nucleic acids of the Nlb gene (see Table 1) was used to estimate the identity value among all the Potyvirus sequences analyzed. To do this, the MatGAT application (Global Alignment Tool atrix) that generates similarity / identity matrices for DNA sequences was used (Campanella JJ, et ai., BMC Bioinformatics. 2003, 4: 29).

Table 1. Preserved regions of the Nlb gene selected from the 94 sequences of

Potyvirus analyzed.

 Virus Reference SEQ ID

 NCBI NO:

Agropyron IMC mosaic virus 005903.1 11

! Virus Algerian watermelon mosaic NC 010736.1 12

CN Y virus 014905.1 13

Arrachacha NC 018176.1 mottle virus 14

Banana bract mosaic virus NC 009745.1 15

Baselia rugged mosaic virus NC 009741.1 16

Common necrotic mosaic virus from bean NC 004047.1 17

Jewish common mosaic virus NC 003397, 1 18

Yellow Bean Mosaic Virus NC 003492.1 19

Beet mosaic virus NC 005304.1 20

Bidens mosaic virus isolated SP01 NC 023014, 1 21

Bidens NC 014325.1 mottle virus 22

Chamaesciíla corymbosa virus NC 019415.1 23

Brugmansia suaveolens NC 014536.1 mottle virus 24

Yellow striatum virus of achira NC 013261.1 25

Celery mosaic virus NC 015393.1 26

Chili ring spots virus NC 016044.1 27

Chilli vein mottle virus NC 005778.1 28

Clover yellow veins virus NC 003536, 1 29 Virus Reference SEQ ID

 NCBI NO:

Dactyl striatum virus NC 003742.1 30

Colombian virus of datura NC 020072, 1 31

Caupi mosaic virus transmitted by aphids NC 004013.1 32

Daphne NC Mosaic Virus 008028.1 33

Malanga NC 003537.1 mosaic virus 34

East Asian Passiflora NC virus 007728.1 35

Freesia NC mosaic virus 014084.1 36

Fritiliaria Virus Y NC 010954.1 37

Habenaria NC mosaic virus 021786.1 38

Hardenbergia NC mosaic virus 015394.2 39

Hippeastrum NC 017967.1 40 mosaic virus

Hordeum NC mosaic virus 005904.1 41

Iranian Sorghum Mosaic Virus from Aiepo NC 018833.1 42

Yam Japanese Mosaic Virus NC 000947.1 43

Sorghum mosaic virus of Aiepo NC 003606.1 44

Keuni ^' orong mosaic virus isolated Cheongwon NC 016159.1 45

Konjac NC Mosaic Virus 007913.1 46

Yellow striated virus from leek NC 00401 1.1 47

Iilium NC 005288.1 mottle virus 48

Lupine mosaic virus NC 014898.1 49

Corn dwarf mosaic virus NC 003377, 1 50

Watermelon Moroccan mosaic virus NC 009995.1 51

Daffodil degeneration virus NC 008824.1 52

Narcissus late yellowing virus isolated NCJ323628.1 53 Marijiniup8

 Yellow daffodil virus of narcissus NC 011541.1 54

Onion yellowing virus of onion NC 005029.1 55

Ornithogaíum NC 019409.1 56 mosaic virus

Panax NC Virus 014252.1 57

Mosaic potyvirus with leaf distortion of NC_005028.1 58 papaya

Papaya ring spot virus NC 001785.1 59 Virus Reference SEQ ID

 NCBI NO:

Fruit hardening virus of passion fruit NC 014790.2 60

Pea mosaic virus transmitted by seeds NC 001671, 1 61

Peanut Spotting Virus NC 002600.1 62

Pennisetum NC 007147.1 mosaic virus 63

Pepper mottle virus NC 001517.1 64

Severe pepper mosaic virus NC 008393.1 65

Pepper yellow mosaic virus RNA strain: NC_014327.1 66 DF

 Peruvian tomato mosaic virus NC 004573.1 67

Phyto lacea mosaic virus isolated PkMV PA NC 018872.2 68

Potato virus A NC 004039.1 69

Potato virus V NC 004010, 1 70

Chive mosaic virus NC 003399.1 71

Yellow striatum virus of the chaiota NC 007433.1 72

Sorghum mosaic virus NC 004035.1 73

Soy mosaic virus NC 002634.1 74

Sugarcane mosaic virus NC 003398.1 75

Chlorotic mottled sunflower virus NC 014038.1 76

Latent sweet potato virus NC 020896.1 77

Sweet potato virus NC 017970, 1 78

Sweet potato virus C 014742.1 79

Sweet potato G virus isolated Jesús Maña NC 018093.1 80

Telosma NC Mosaic Virus 009742, 1 81

Thunberg fritiiiary NC 007180.1 82 virus

Mosaic virus with tobacco vein banded NC 009994.1 83

Speckling virus of tobacco veins NC 001768.1 84

Tomato necrotic dwarf virus NC 017824.1 85

Turnip mosaic virus NC 002509.2 86

Valiota speciosa virus NC 017977.1 87

Virus Y of verbena NC 010735.1 88

Wild Potato Mosaic Virus NC 004426, 1 89

Wild tomato mosaic virus NC 009744.1 90 Virus Reference SEQ ID

 NCBI NO:

Wisteria veins mosaic virus NC 007216.1 91

Mosaic virus of the ^' bed NC 016441, 1 92

Brazil Soft Yam Mosaic Virus NC 019412.1 93

Yam mosaic virus NC 004752.1 94

Creek soft mosaic virus NC 011560.1 95

Zucchini tabby mosaic virus isolated NGJ323175.1 96 Re01 25

 Zucchini yellow mosaic virus NC 003224.1 97

Lettuce mosaic virus NC 003605.1 112

Pepper vein mottle virus NC 011918.1 113

Sharka disease virus NC 001445.1 114

Potato virus Y NC 001616, 1 115

Sweet spotted sweet potato virus NC 001841.1 116

Tobacco Engraving Virus NC 001555.1 117

Watermelon mosaic virus NC 006262.1 118

Plant materials

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., Plañí. 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 Viroíogicaí Methods. 1999, 82: 167-175.). In summary, 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 μΙ) on nylon membranes. Synthesis of cDNA clones

 Reverse transcription and PCR reactions were performed as previously described (Herranz M. C, et al, Journal oí Vimíogicai 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. The incorporation of the purified PCR fragment into the pSK + plasmid in the correct orientation allows the inactivation of the original Xhoi site, present in the pSK + plasmid, by the compatible Salí site present at the 3 'end of the PCR product. This allows the use of! new Xhoi site, present in e! 5 'end of! PCR product, for the synthesis of the ribosonde or the incorporation of a new PCR fragment (Peiro A., et al., European Journal heard Plani Pathology. 2012, 132: 469-475).

Using this strategy, seven cDNA fragments were introduced into e! plasmid pBluescript SK (+) corresponding to the clones of LMV (SEQ ID NO: 7), WMV (SEQ ID NO: 6), PVY (SEQ ID NO: 5), PVMV (SEQ ID NO: 4), PPV (SEQ ID NO: 3), SPFMV (SEQ ID NO: 2) and TEV (SEQ ID NO: 1). Seven simple gender probes were generated comprising SEQ ID NO: 1 or 2, or 3, or 4, or 5, or 8 or 7 each. In addition, three gender probes (GP) were generated that differed in the number of pathogen sequences incorporated. Thus, GP3 (SEQ ID NO: 8), GP5 (SEQ ID NO: 9) and GP7 (SEQ ID NO: 10) 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).

Table 2. Pair of primers used in the amplification of gender probes.

Sequence SEQ ID Fragment H ° of access polynucleot dica (5 ^' -3 ^' ) ^to NO: expected NCBI and viral

Cpb target) localization in genome ^b

CACACTCGCGGTAAGT 98 LMV 496 X97704 TTGGAGTGTGGAA 7961-8457 Fragment

 viral

 Diana

CACAGTCGACGTCATA 99 LMV 496 X97704 GCTAGCACAACCAT 7961-8457

CACACTCGAGAAGGG WMV EU660589 A ATTTG G A AYG GTTC 7882-8348

CACAGTCGACTTGTCA 101 WMV EU660589 ACGACTGTAGATGG 7882-8348

CACACTCGAGATTTGG 102 PVY AJ890346 AACGGATCATTGAA 7510-7982

CACAGTCGACACCATG 103 PVY AJ890346 AGAGAATTATCCAC 7510-7982

CACACTCGAGGTAAAC DQ645484 TTGGGATTTGG 7530-8009

CACAGTCGACCATGAG 105 DQ645484 AGTGTTATC 7530-8009

CACACTCGAGGAAAGA PPV KU508427

AAGGAGTGTGGAATG 7495-7993

G

CACAGTCGCGTCATTG 107 PPV KU508427 CCAAAATAACCAT 7495-7993

CACACTCGAGATGGGA SPFMV KU511268 TAC A A AG GTCTYTG G A 8563-9056 A

CACAGTCGACGCTAAC SPFMV 493 KU5 1268 ACAACCATAAGTGT 8563-9056

CACACTCGAGGAAAGC 1 1 ⁽ TEV DQ986288 TGGGAATTTGG 7468-7947

CACAGTCGACCATGAG 1 11 TEV DQ986288 TGTGTTGTC 7468-7947 ^a The restriction sites of Xhol and Sañ are underlined,

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

For the synthesis of the gender probes, 1 pg of was linearized! corresponding plasmid with the restriction enzyme Xhol, was purified by extraction with phenol-chloroform and precipitated with ethanoi. The linearized plasmid was used to synthesize the gender probe as previously described (Mas P., et al., Journal o Virological Methods. 1993, 45: 93-102; Pallás V., et al., Methods in Molecular Biology 1998, 81: 481-468). 1 μΙ of the total nucleic acids extracted from the leaf tissue by the method of the silica method (undiluted or of the corresponding dilutions) was applied directly on positively charged nylon membranes (Roche Diagnostics GmbH, anheim, Germany), dried at air and were covalently fixed to the membrane by irradiation with ultraviolet (UV) light (700 ^ 100 Ü / cm ² ), Prehybridizations and hybridizations were performed with the gender probes as previously described, with the only difference in the selected temperature for hybridization (Pallás V ,, et al., Methods in Molecular Biology. 1998, 81: 461-468; Sánchez-Navarro JA, et ai, Plant Pathology. 1998, 47: 780-786). All gender probes were used at the same concentration in the hybridization solution (20 ng / ml). Chemiluminescent detection using CDP-Star® reagent as a substrate was performed as recommended by the manufacturer (Roche Diagnostics GmbH, Manheim, Germany). The films were exposed for 30 minutes.

Estimation of the detection limit of the hybridization assays

For the estimation of the detection limit of the individual or single probes (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) or the three gender probes (SEQ ID NO: 8, 9 or 10), 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.

The ability of 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. To do this, 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 / μΙ. In addition, total 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. However, the eight degree reduction during the hybridization test (80 ° C) was sufficient to detect cross hybridization with the TEV sequence (SEQ ID NO: 1) at high concentration (200 pg / μΙ), while the reduction of another five (55 ° C) or ten (50 ° C) degrees was sufficient to detect two (TEV, SPFMV) or all sequences at different concentrations (Fig. 2A). Hybridization performed at 50 ° C has also shown a weak signal in the negative controls (total RNA extracted from healthy tissue). The result shown in Fig. 2A is summarized in Fig. 2B, in which the numbers represent the percentage of identity between the PPV sequence (SEQ ID NO: 3) and the rest of the Potyvirus sequences, while the Gray colors indicate the last number of transcripts that showed hybridization with the PPV probe (SEQ ID NO: 3). The corresponding picograms (pg) of transcript assigned for each gray color are indicated on the right side of Fig. 2B. White indicates absence of hybridization signal. In the next stage, cross hybridization of the gender probes at 55 ° C was evaluated (Fig. 2C). The data shows that, in general, 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.

Subsequently, the cross-reactivity of the three gender probes of the present invention (GP3, GP5 and GP7) was evaluated (Fig. 3). For this purpose, replicas of the same membrane described in Fig. 2 were used which hybridized overnight at 50 ° C, since no cross hybridization was observed with the negative controls at this temperature. GP3, GP5 and GP7 were at 20 ng / ml in the hybridization solution and the films were exposed for 30 minutes. First, it was observed that the shorter genus probe, GP3 that carried only three tandem sequences (SEQ ID NO: 3 (PPV), SEQ ID NO: 2 (SPFMV) and SEQ ID! MO: 1 (TEV) 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 / μΙ (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. Thus, a positive hybridization signal was observed for all viral sequences included in GP5 up to a concentration of 0.06 pg / μΙ, while the other two sequences not present in these gender probes were detected up to a concentration of 1 , 6 pg / μΙ, with a sensitivity five times lower than GP3 (SEQ ID NO: 8).

Finally, 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 / μΙ. As can be seen in the case of 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. Fig. 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.

The results obtained in 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. To identify how many Potyvirus sequences could hybridize potently with the three genus probes of the invention (GP3, GP5 and GP7), 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). The results, summarized in Table 3, showed that all Potyvirus strains had an identity percentage of 68% or higher with two or more of the seven selected sequences 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. Only the onion dwarf yellowing virus, with a percentage of 69.3%, showed that identity only with PPV (SEQ ID NO: 3). In addition, most of the sequences had identity percentages of 70% or higher with any of the selected sequences. Only Daphne mosaic virus, Habenaria mosaic virus, Hordeum mosaic virus, onion dwarf yellowing virus, Ornithogaium mosaic virus and Valiota speciosa virus presented identity percentages below 70% In addition, the 94 strains of Potyvirus could theoretically be detected with the lowest GP3 genus probe, since all sequences had a percentage of identity greater than 68% with any of the cloned fragments of PPV (SEQ ID NO: 3), TEV (SEQ ID NO: 1) and SPFIV1V (SEQ ID NO: 2).

Table 3. Percentage of identity estimated by MatGAT between the sequences used for the individual or single gender probes of LV (SEQ ID NO: 7), PPV (SEQ ID NO: 3), PVMV (SEQ ID NO: 4), PVY (SEQ ID NO: 5), SPF V (SEQ ID NO: 2), TEV (SEQ ¡D NO: 1), WV (SEQ ID NO: 6) and the equivalent sequence of the indicated Potyvirus. Identity percentage values of 68% and 69% are marked in light gray, while values of 70% and higher are marked in dark gray.

Finally, the ability of the three GP3, GP5 and GP7 genus probes to detect heterologous Potyvírus was analyzed by analyzing 43 different field samples including different hosts, with respect to the presence of 32 Potyvírus strains (Table 4). All samples were obtained from the German collection of microorganisms and cell cultures (DSMZ) or the Mediterranean Agroforestry Institute of the Polytechnic University of Valencia. Samples were extracted using the silica protocol (acKenzie DJ, et al., Piant Disease. 1997, 81: 222-226) but also with a rapid protocol in which the tissue is homogenized with citrate buffer and applied directly to the Membrane (Sánchez-Navarro et al ,, Píant Pathology. 1998, 47: 780-786; Sánchez-Navarro JA, et a!., Journal of Virologicai Methods. 1999, 82: 167-175). In the two extraction protocols, the amount of tissue applied to the membrane corresponded to 0.2-0.3 mg. Hybridizations were performed overnight at 50 ° C with a gender probe concentration of 20 ng / mi and the films were exposed for 30 minutes. All results are summarized in Table 4. First, no hybridization signal was observed with any of the 8 healthy hosts analyzed, although hybridization was performed at 50 ° C. The 32 Potyviruses were detected with GP3, GP5 and GP7, except in the case of PVMV (SEQ ID NO: 4), which gave a negative signal with GP3. It is interesting to note that 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). On the other hand, it was observed that 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.

In addition, when the plants were analyzed using the rapid citrate buffer protocol, it was observed that 89.9% of the positive ones on silica (80 of 89) were correctly detected. Two samples were also observed that were positive by citrate buffer extraction and negative by silica extraction, using the GP3 and GP5 gender probes (samples 30 and 34, Table 4). These results could indicate differences in the viral titer associated with the extraction protocol used, but other effects derived from the starting material or the host (Nicotiana benthamiana or Cucúrbita pepo) that could interfere with the detection procedure cannot be discarded.

Table 4. Analysis of the ability of the GP3, GP5 and GP7 genus probes to detect different Potyviruses in field samples by point nucleic acid hybridization (NASH). All samples were extracted with silica and citrate buffer protocols. Hybridization was performed at 50 ° C.

NASH

Virus Source ^to Source Hospedador

(Silica / ¹ citrate buffer)

GP3 and GP5 GP7

Virus Y from DSMZ

 Australia Daucus carota + / + + / + carrot (CarVY) (PV0759)

 ! Virus

DSMZ yellowing

 Germany Ailium strain +/- +/- + / + dwarf of the (PV0447)

onion (OYDV)

 Mosaic Virus

 UPV Spain Lactuca sativa + / + +/- turnip (Tu V)

 Mosaic Virus

 DSMZ Triticum

from Agropyron Germany + / + + / + + / +

 (PV0729) aestivum

(AgMV) ASf- i

Source ³ Virus Source Host

 {Sñlcí ϊ / Citrate buffer)

GP3 GP5 GP7

Mosaic Virus

 common necrotic DSMZ Countries Phaseolus + / + + / + + / + of ia Jewish (PV0413 Low vulgaris

Mosaic Virus

Mosaic Virus

Mosaic Virus

! Virus fluted

 ! Virus mottled

 Nicotian DSMZ

 from! Pepper Morocco + / * + / + + / +

 (PV0256) tabacum

 (PepMoV)

 ! Virus mosaic

 Nicotian DSMZ

Peruvian of! Peru + / * + / + + / +

 (PV0399 tabacum

 tomato (PTV)

 Virus A from! To DSMZ Nicotiana

 Germany + / + + / + potato (PVA) ÍPV0535) western! s

 Nicotiana DSMZ V virus

 South America + / + + / + + / + potato (PW) (PV0319) Benthamiana

 Nicotiana DSMZ Y virus

 Germany + / * + / + +/- potato fPVY) (PV0343) tabacum

 ! Virus Nicotian DSMZ mosaic

China + / + · ^A soybean (SMV) (PV0938) Benthamian

 Mosaic Virus

 with DSMZ Nicotiana banded

 China + / + + / + + / + veins of! tobacco (PV0721) benthamiana

Mosaic Virus

Mosaic Virus

 Virus Y of! celery DSMZ

 Germany Ami majus + / + + / + + / + (ApVY) (PV1001)

 Mosaic Virus

 Yellow of the DSMZ Nicotiana

 South America + / * + / + + / + pepper (PV0026 tabacum

(PepYMV) NAS 1

Source ³ Virus Source Host

 (Silica ϊ / Citrate buffer)

GP3 GP5 GP7

Nicotiana DSMZ virus 2 - / - + / + + / + sweet potato (SPV-2) (PV1036) benthamiana

 Mosaic Virus

 Nicotian DSMZ

 Germany yellow + / + + / + + / +

 (PV0717) Benthamiana

Jewish (BYMV)

 Leaf virus n¾M7

 fine from Germany Daucus carota + / + + / + + / +

 (PV1005)

 carrot (CTLV)

 Apium mosaic virus

 UPV Spain + / nf + / nf + / nf celery (Ce V) graveo lens

 Vein virus

 DSMZ Phaseolus

 yellow from Germany + / nf + / nf + / nf

 (PV0367) vulgaris

 clover (CYYW)

 Mosaic Virus

 of Freesia UPV Spain Freesia + / + + / + +/-

(Fre V)

 Virus

 ring spots States

 UPV Ciirulius ianatus + / nf + / nf + / nf of Papaya United

 (PRSV)

 Engraving virus DSMZ States Nicotiana + / +

 of tobacco (TEV) (PV0308) United tabacum

 Etching virus Physalis HH / HH + / + + / + of floridana tobacco (TEV)

Mosaic Virus

 Spotted virus

 Nicotian DSMZ

 of the veins of Ghana + / + + / +

 (PV0257) Benthamiana

pepper (PV V)

 Virus Y of the

 UPV Spain „/ - + / + + / + potato (PVY)

 Spotted virus

 Nicotian DSMZ

 soft sweet potato Kenya + / + + / +

 (PV0898) Benthamiana

 (SPFMV)

 Engraving virus Nicotian States

 + / + + / + + / + of tobacco (TEV) United tabacum

Mosaic Virus

 Sharka Prunus virus

 Lab Spain + / nf + / nf + / nf (PPV) domestica

 Healthy control Lab Spain Cucumis meló - / nf - / nf - / nf

 Cucumis

 Healthy control Lab Spain - / nf - / nf - / nf sativus

 Nicotiana

Healthy control Lab Spain _ /. _ /. - / - Benthamiana ASf- i

Source ³ Virus Source Host

 (YES ICON ϊ / Citrate buffer)

GP3 GP5 GP7

SoÍanum

 Healthy control Lab Spain ~ / nf - / nf - / nf lycopersicum

 Chrysanthemu

 Healthy control Lab Spain - / nf - / nf - / nf m

 Gynura

 Healthy control Lab Spain - / nf - / nf - / nf

 auraniac

 Nicotiana

 Healthy control Lab Spain _ /. ./. ./_ tabacum

 Arabidopsis

 Sound control UPV Spain ./. - / - _ /.

 íhaíiana

 +, - and * correspond to the positive, negative and doubtful results, respectively, nf, fresh tissue is not available.

a, samples obtained from the Isabel Font group at the Polytechnic University of Valencia (UPV), the German Collection of Microorganisms and Cell Cultures (DSMZ) or our laboratory (Lab).

Claims

one . An isolated polynucleotide sequence comprising, arranged in tandem, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.  2. The polynucleotide according to claim 1 comprising SEQ ID NO: 8.  3. The polynucleotide according to any one of claims 1 to 2, consisting of SEQ ID NO: 8.  4. The polynucleotide sequence according to any one of claims 1 to 3, further comprising at least two of the nucleotide sequences selected from the list consisting of: SEQ ID NO: 4, 5, 6, 7 and / or Any combination thereof.  5. The polynucleotide sequence according to claim 4, wherein the nucleotide sequence comprises SEQ ID NO: 4 and 5, or SEQ ID NO: 4, 5, 6 and 7.  6. The polynucleotide sequence according to any one of claims 1 to 5, comprising SEQ ID NO: 9 or SEQ ID NO: 10. 7. The polynucleotide sequence according to any one of claims 1 to 6, consisting of SEQ ID NO: 9 or SEQ ID NO: 10. 8. The polynucleotide sequence according to any one of claims 1 to 7 characterized in that it is a probe.  9. In vitro use of a polynucleotide sequence according to any one of claims 1 to 8, to detect and / or quantify viruses belonging to the genus Potyvirus in a single assay.  10. In vitro use according to claim 9, wherein the detection and / or quantification is performed in a single isolated sample.  eleven . In vitro use according to claim 10, wherein the sample is a vegetable or a plant sample.  12. Method for the detection and / or quantification in vitro of the complete series of viruses belonging to the genus Potyvirus in a test sample, comprising the steps of:  a) obtain a total nucleic acid preparation from the test sample, b) contacting the stage I nucleic acid preparation prior, with the polynucleotide according to any of claims 1 to 8,  c) detecting hybridization between the polynucleotide according to any one of claims 1 to 8 and the nucleic acid of the previous step, where hybridization indicates that the test sample comprises a Potyvirus. 13. The method according to claim 12 wherein, alternatively, in step a) the test sample can be homogenized with a lamp. 14. A method according to any one of claims 12 to 13, wherein the contact of the test sample with the polynucleotide according to claims 1 to 8 comprises point transfer hybridization. 15. A method according to any one of claims 12 to 14, wherein the polynucleotide according to claim 1 to 8 is immobilized with a nylon membrane.  16. The method according to claim 15, wherein the immobilization is carried out by temperature or by UV light. 17. Method according to any one of claims 12 to 16, wherein the hybridization is performed at a temperature of at least 50 ⁹ C. 18. A method according to any one of claims 12 to 17, wherein the test sample is a vegetable or a plant sample. 19. Method according to any one of claims 12 to 18, wherein the Potyviruses are selected from the list consisting of: Agropyron mosaic virus, watermelon Algerian mosaic virus, Y celery virus, celery virus mottled of the arracacha, banana bract mosaic virus, basella rugged mosaic virus, common necrotic mosaic virus of the bean, common bean mosaic virus, yellow bean mosaic virus, mosaic virus beet, Bidens mosaic virus, Bidens mottle virus, Chamaescilia corymbosa virus A, Brugmansia suaveolens mottle virus, yellow achira striatum virus, celery mosaic virus, chili ring spot virus, chili vein mottle virus, clover yellow vein virus, dactyl striatum virus, Colombian datura virus, aphid-transmitted cowpea mosaic virus, Daphne mosaic virus, vi malanga mosaic rus, dei virus this Asian of Passifiora, Freesia mosaic virus, FritiHaria virus Y, virus! Habenaria mosaic, virus! Hardenbergia mosaic, virus! Hippeastrum mosaic, virus! Mosaic of Hordeum, virus! Iranian mosaic of! sorghum of A! epo, virus of! Japanese mosaic of! yam, virus! mosaic of! Aiepo sorghum, virus! mosaic of! Keunjorong, virus! mosaic of! Konjac, leek yellow striatum virus, lettuce mosaic virus, iiiium mottle virus,! mosaic of! lupine, dwarf mosaic virus from! Corn virus! Moroccan mosaic of watermelon, degeneration virus of! Daffodil virus! late yellowing of daffodil, virus! yellow striated daffodil, dwarf onion yellowing virus, Ornithogalum mosaic virus, Panax virus Y, virus! Mosaic with papaya leaf distortion, papaya ring spot virus, hardening fruit virus! passion fruit, pea mosaic virus transmitted by seeds, peanut mottle virus, Pennisetum mosaic virus, pepper mottle virus, severe mosaic virus from! pepper, pepper vein mottle virus, pepper yellow mosaic virus, Peruvian mosaic virus from! tomato, sharka disease virus, phytolacca mosaic virus, potato virus A, potato virus V, potato virus Y, chive mosaic virus, virus! yellow striated shallot, virus! mosaic of! sorghum, soy mosaic virus, virus! Sugarcane mosaic, virus! chlorotic mottled of! sunflower, sweet potato sweet spotted virus, latent sweet potato virus, sweet potato virus 2, sweet potato virus C, sweet potato G virus, virus! Telosma mosaic, Thunberg íritiüary virus, virus! Engraving of! tobacco, virus! mosaic with banded veins of! tobacco, tobacco vein mottling virus, tomato necrotic dwarf virus, turnip mosaic virus, Vailota speciosa virus, verbena virus Y, watermelon mosaic virus, virus! Mosaic of the wild potato, virus! mosaic of! wild tomato, virus! mosaic of wisteria veins, virus! Jicama mosaic, soft yam mosaic virus, yam mosaic virus, soft creek mosaic virus, virus! tabby mosaic of! Zucchini and virus! Zucchini yellow mosaic. 20. Kit or matrix to detect and / or quantify viruses belonging to the genus Potyvirus comprising the polynucleotide sequence according to a any one of claims 1 to 8, in an isolated sample.  twenty-one . Kit or matrix according to claim 20, wherein the sample is a vegetable or a plant sample.  22. Use of the kit or matrix according to claim 21 to detect and / or quantify viruses belonging to the genus Potyvirus in an isolated sample. 23. Use according to claim 22, wherein the sample is a vegetable or a plant sample.