ES2205998B1 - PROCEDURE FOR THE DETECTION OF THE VIRUS OF THE YELLOW OF THE CUCUMBER VEINS (CVYV). - Google Patents

Abstract

Procedure for the detection of the cucumber vein yellow virus (CVYV). The object of the present invention is a specific, sensitive and rapid procedure for the detection of Cucumber vein yellowing virus (CVYV) in plants. The method comprises the preparation of a genetic construct containing a fragment of the CVYV genome, the preparation of a nucleic acid probe from said genetic construction and the hybridization of said probe with nucleic acid extracts or with imprints of tissue sections of the plants to which the procedure applies.

Description

Procedure for the detection of the virus yellowing of cucumber veins (CVYV).

Technical sector

Biotechnology. Molecular Hybridization Techniques for viral detection.

The object of the present invention is a specific, sensitive and rapid procedure for the detection of Cucumber vein yellowing virus (CVYV) in plants. Furthermore, the present invention is about the genetic construction used in said method.

State of the art

CVYV is the cause of serious epidemics in cucumber and other cucurbit crops in the eastern Mediterranean basin, causing major economic losses. CVYV has rod-shaped particles 740-800 nm in length and 15-18 nm in diameter (Sela et al., 1980). The virus is transmitted semi-persistently by the whitefly Bemisia tabaci and can also be transmitted by mechanical inoculation. The range of CVYV guests is restricted to the Cucurbitaceae family (Mansour and Al-Musa, 1993). Two virus isolates from Israel (CVYV-Is) and Jordan (CVYV-Jor) have recently been characterized (Lecoq et al., 2000). A cytological analysis of hosts infected by these two isolates has shown the appearance of typical formations of the virus infection of the Potiviridae family and, therefore, it has been proposed that CVYV belongs to said family. The characterization of a nucleotide sequence corresponding to a fragment of the CVYV-Is genome has confirmed this idea (Lecoq et al. 2000). Thus, CVYV appears to be a member of the genus Ipomovirus , within the Potiviridae family, and like other members of this family it must have a single-stranded and messenger RNA genome. However, the complete CVYV genome sequence is still unknown.

CVYV has been observed for the first time in Spain in the fall of 2000 in protected crops of cucumber from Almeria (Cuadrado et al., 2001). From this moment the virus has spread very quickly in this area, first in the crops of cucumber, and then to crops of other cucurbitaceae. Only in this area the economic losses caused by CVYV until the date have been very large and therefore the design and use of virus control strategies that are effective. The control of the virus by controlling its vector is a strategy that has already been revealed insufficient in other cases of whitefly transmitted viruses, such that it is The use of alternative strategies is essential. Among these are It has the management of certain cultural practices such as use of virus-free propagation material and the use of genetically resistant virus cultivars (Matthews, 1991). In in any case, for the implementation of these strategies of control is essential to have detection methods of Viruses that are specific, sensitive and fast. The existence of detection methods with these features can facilitate hugely processes like sanitary material certification plant or virus susceptibility analysis of entries Cucurbitaceae germplasm candidates to be carriers of genetic resistance to CVYV. Thus, the objective of this invention is provide a CVYV detection method that is specific, sensitive and fast. There are numerous methods of virus detection of plants. Among the most widely used are those based on serological techniques, particularly those based on the methodology ELISA (Matthews, 1991). For the operation of this type of methods it is essential to have specific antibodies against the virus In the case of CVYV they have not yet been able to get these antibodies, probably because of the difficulty that It involves the purification of viral particles. An alternative to Serological techniques consist in the use of techniques based on molecular hybridization. For virus detection by molecular hybridization is essential to have constructions genetics containing nucleotide sequences homologous to viral genome, since from these constructions are synthesized the probes that are used to indicate the presence or absence of virus in plant samples. Virus detection by molecular hybridization has been widely used, in particular hybridization over total nucleic acid extracts of plants applied on nitrocellulose or nylon membranes (Matthews, 1991). However, the preparation of extracts from plant nucleic acids require some work, and that's why recently hybridization techniques have been developed molecular on imprints of tissue sections of plants whose obtaining is simple (see for an example Narváez et al., 2000). Thus, the objective of the present invention is to provide a CVYV detection method based on hybridization molecular. This method must be functional on acid extracts plant nuclei and on imprints of tissue sections of plants.

Explanation of the invention.

The object of the present invention is a method for the detection of Cucumber vein yellowing virus (CVYV) in cucurbits by molecular hybridization techniques. The procedure is applied to the detection of CVYV, among other plants, in cucumber, melon and zucchini and includes the following steps:

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preparation of a genetic construction which contains a fragment of the CVYV genome.

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preparation of an acid probe nucleic acids from the plasmid obtained in the step previous.

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probe hybridization with extracts of nucleic acids or with imprints of tissue sections of the plants to which the procedure applies.

For the preparation of genetic construction which contains a fragment of the CVYV genome two are selected oligonucleotides from the CVYV sequence:

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he oligonucleotide that is used for the synthesis of the first chain  of the cDNA is the 5'-ACATTAGCGGCAAGTGTCTG-3 '(MA163, SEQ ID NO 1) or any complementary oligonucleotide in at least 95% of its nucleotides to the sequence of CVYV-AILM (SEQ ID NO 3).

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he oligonucleotide used in the construction of the second chain of the cDNA is the 5'-GTCAGCCGTCAACTGTGGTGG-3 '(MA162, SEQ ID NO 2) or any oligonucleotide included in the sequence of nucleotides of CVYV-AILM (SEQ ID NO 3) and have with it a similarity of at least 95%.

The nucleotide sequence thus obtained and described by the present invention and belonging to the viral isolate CVYV-AILM (SEQ ID NO 3) is part of this invention. The double chain of cDNA obtained through the use of oligonucleotides undergo ligation and subsequent insertion into a expression vector, a plasmid among others, from the which can be synthesized the probes that will be used subsequently in the identification of the presence or not of virus in plant samples.

In the present invention the construction of plasmid pLMCVYV, which contains the sequence SEQ ID NO 3, and is part of the present invention.

For the development of the detection method of CVYV in the present invention an RNA probe was obtained Complementary (cRNA) to viral RNA for its best performance with compared to other types of probes (Matthews, 1991) as of plasmid pLMCVYV.

The results, described herein invention of a CVYV detection by hybridization with the previously mentioned probe (cRNA) showed that signal appeared only in those samples from plants that were infected with CVYV, while in healthy plants and in plants infected by viruses other than CVYV did not appear signal (Fig. 2). That is, the detection method is specific to CVYV. In addition, the results obtained in the present invention indicate that the sensitivity of this CVYV detection method is 6 pg (Fig. 3).

On the other hand, this detection procedure of CVYV by hybridization in cucurbitaceae plants was performed, in the present invention, on nucleic acid extracts plant totals and on imprints of tissue sections of plants - in this last case the obtaining is much simpler - observing that said detection is specific to CVYV.

Brief description of the figures

Figure one

Situation in the genome of potyvirus of the genome fragment of CVYV-AILM that has been cloned and sequenced to this invention

Under the diagrammatic representation of the genome of potyvirus appears a gray segment (LMAM48) that indicates the size (approximately 1.5 kbp) and the situation in the genome (comprising part of the NIb and CP genes) of the fragment cloned

Figure 2

Result of a membrane hybridization illustrating the specificity of the detection method

At the top of the figure appears indicated the origin of the samples that were included in the membrane as for the host and the virus with which he had been inoculated (or not, for healthy controls). To the right of the figure is indicated the type of material that was included in the membrane, either tissue imprints or acid extracts nucleic

Figure 3

Result of a membrane hybridization illustrating the detection method sensitivity

At the top of the figure appears indicated the amount of transcribed viral RNA (tRNA) that was included in each sample (or not, for negative controls). In the right part of the figure shows the RNAt diluent which was used in each treatment.

Detailed description of the invention

The development of the invention has involved the steps listed below. For the techniques of Molecular Biology has essentially been followed by Sambrook et al. (1989).

Biological cloning of a CVYV isolate

During the autumn of 2000 crops were sampled Cucumber protected in the province of Almeria. In a certain number of greenhouses were detected plants with symptoms Characteristics of CVYV infection: intense yellowing of veins and chlorosis in young leaves and mottled chlorotics in fruits. With these samples were mechanically inoculated (Mathews, 1991) 3 seedlings of each of the following species: melon, cucumber and zucchini. These plants were kept in a culture chamber with day / night light cycle of 16/8 hours and temperatures of 24 ° C day and 19 ° C night. 10-15 days post-inoculation reproduction of the symptoms of chlorosis and intense yellowing of leaf veins in 2 Melon plants, no cucumber and 3 zucchini. But nevertheless, the presence of CVYV in the two melon plants could be verified symptomatic and also in all other plants (symptomatic or no) by an RT-PCR assay (Square et al. 2000). From one of the CVYV infected melon plants a virus transmission was performed using whitefly (López-Sesé and Gómez-Guillamón, 2000). The objective of this essay was to obtain an isolate of CVYV that did not contain viruses other than CVYV that were of mechanical transmission This transmission was carried out using 20, 10 and 5 flies per plant. In the inoculation treatment with 5 flies became infected only one of the 5 inoculated plants. This suggested that the multiplicity of the infection in this treatment was low, and therefore this plant has been taken as CVYV clonal inoculum source (Matthews, 1991). To the isolated viral originating from this plant has been called CVYV-A1LM.

Preparation of a genetic construct that contains a CVYV-AILM genome fragment

For the molecular cloning of a complementary DNA (cDNA) to a fragment of the virus genome, oligonucleotide primers were designed from the CVYV-Is sequence published by Lecoq et al. (2000) that were used in an RT-PCR experiment. The oligonucleotide for the synthesis of the first cDNA chain was 5 'ACATTAGCGGCAAGTGTCTG 3' (MA163, SEQ ID NO 1), complementary to nucleotides 1531 to 1550 in the sequence of Lecoq et al. (2000). This sequence is found in the 3 'terminal third of the cistron that encodes the capsid protein (CP) of the virus (Fig. 1). The oligonucleotide used in the construction of the second cDNA chain was 5'GTCAGCCGTCAACTGTGGTGG3 '(MA162, SEQ ID NO 2), identical to nucleotides 14 to 34 in the sequence of Lecoq et al. (2000). This second sequence is found in the cistron that encodes the NIb protein of the virus (Fig. 1). As a template for cDNA synthesis, an RNA extract from CVYV-A1LM infected plants prepared using the TRI-reagent reagent (Sigma Chemical Co., St. Louis, MO, USA) was used following the manufacturer's instructions. The synthesis of the first cDNA chain was performed using 10 µL of RNA from infected plants at a concentration of 1 µg / µl. To this RNA was added 1 µL of 0.1 M methyl mercury hydroxide and kept at room temperature 10 min. The following reagents were then added: 1 µl of 1.4 M β-mercaptoethanol, 5 µl of reverse transcriptase buffer concentrated 5 times (Boehringer Mannheim, Mannheim, Germany), 1 µl of a mixture of 10mM dNTPs , 50 units of RNase inhibitor (Amersham Pharmacia Biotech, UK), 1 µL of 100 mM DTT, 1 µl of reverse transcriptase (Expand RT, Boehringer) and 2 µl of MA163 oligonucleotide (SEQ ID NO 1) to one concentration of 100 ng / µl, all in a final volume of 25 µl. This mixture was maintained at 37 ° C for 1 h. Then the second chain of the cDNA was synthesized. To 1 µl of the above mixture was added 5 µl of Taq polymerase buffer 10 times concentrated (Bioline, London, UK), 2 µL of 25 mM Cl 2 Mg, 1 µl of 10 mM dNTPs, 0 , 5 µl of the oligonucleotides MA162 (SEQ ID NO 2) and MA163 (SEQ ID NO 1) at a concentration of 100 ng / µl and 1 µl of Taq polymerase (BioTaq; Bioline), all in a final volume 50 \ mul. This mixture was subjected to the following PCR cycles: one cycle of denaturation at 94 ° C for 5 min, thirty cycles of denaturation at 94 ° C for 30 s followed by hybridization of the oligonucleotides at 52 ° C for 30 s followed by polymerization of the cDNA at 72 ° C for 1 min, and a final polymerization cycle at 72 ° C for 7 min. The PCR result was checked by electrophoresis of an aliquot on an agarose gel stained with ethidium bromide. After transillumination with ultraviolet light, in this gel a band of the expected size for the CVYV cDNA was observed (1.5 kbp; Fig. 1). This cDNA was purified by extraction of a preparative agarose gel that had undergone electrophoresis. Extraction was performed using an Ultrafree-DA column (Millipore, Bedford, MA, USA). The cDNA thus prepared was subjected to a standard ligation reaction using the pGEM-T vector System II kit (Promega, Madison, WI, USA). An aliquot of the ligation reaction was used to transform E. coli DH5α cells. The transformants were analyzed to determine the presence of a 1.5 kbp insert and two transforming colonies with an insert of this size were selected. Plasmid DNA was prepared from both colonies that was used to sequence the 1.5 kbp insert in its entirety for the two colonies of transforming bacteria. Both sequences were identical to each other (SEQ ID NO 3) and very similar (95% nucleotide sequence homology) to the published CVYV-Is sequence (Lecoq et al., 2000). The plasmid containing this insert has been designated pLMCVYV and is part of the present invention.

The nucleotide sequence obtained for CVYV-AlLM can be translated into amino acids uninterruptedly in the reading frame +3. An analysis comparative with sequences of other potyviruses has revealed that this sequence (SEQ ID NO 3, LMAM48) comprises a part 3'-terminal of the cistron encoding NIb of the virus, and a 5'-terminal part of the cistron that encodes the CP of the virus (Fig. 1). In the NIb cistron region there are 26 nucleotide substitutions with respect to the sequence of CVYV-Is. These nucleotide substitutions result in 2 amino acid substitutions in the corresponding protein. Also, in the region of the cistron of the CP there are 47 nucleotide substitutions with respect to the sequence of CVYV-Is resulting in 9 substitutions amino acids. The nucleotide sequence thus obtained and described by the present invention and belonging to the viral isolate CVYV-AILM (SEQ ID NO 3) is part of this invention.

Synthesis of probes from pLMCVYV

For the development of the CVYV detection method, it was decided to prepare complementary RNA probes (cRNA) to the viral RNA for its better performance compared to other types of probes (Matthews, 1991). To prepare these probes, we proceeded as described below. Plasmid pLMCVYV was linearized using the restriction enzyme SphI . For the in vitro transcription reaction, 10 µl of this linearized DNA was used at a concentration of 100 ng / µm to which 20 µl of 5-fold concentrated transcription buffer (Amersham Pharmacia Biotech), 10 µl of 100 mM DTT, 20 pi of a mixture of 2.5 mM rNTPs containing DIG-11-UTP (Boehringer) in a 1 mM concentration, 50 Units of RNase inhibitor (Amersham Pharmacia Biotech) and 50 Units of SP6 transcriptase ( Amersham Pharmacia Biotech), all in a final volume of 100 µl. This mixture was kept at 37 ° C for 1 h 30 min. After this time, 10 units of RNase-free DNase (Boehringer) were added and incubation was continued for another 30 min. Then 4 µl of 0.5 M EDTA, 5 µL of 8 M ClLi and 300 µl of pure ethanol were added to the mixture, kept at -20 ° C for 3 h and the precipitated cRNA was concentrated by centrifugation. The dried precipitate was resuspended in 50 µl of water to which another 50 µl of carbonate buffer pH = 10.2 was added to proceed with controlled hydrolysis of the cRNA. This mixture was maintained at 60 ° C for 56 minutes and then 5 µl of 10% acetic acid, 10 µl of 3 M sodium acetate and 250 µl of pure ethanol were added. It was maintained at -20 ° C for 3 h and the precipitated cRNA was concentrated by centrifugation. The dried precipitate was resuspended in 100 µL of 50% formamide.

CVYV detection by hybridization in acid extracts plant nuclei

For the preparation of acid extracts Plant nuclei proceeded as follows. A piece of foil 0.2 g foliar was introduced into an eppendorf tube, frozen by Immersion in liquid nitrogen and ground with the help of a buril. To the finely ground tissue 400 µl of buffer was added extraction (2% SDS, 100 mM Tris-HCl pH = 8.0, 10 mM EDTA) and the tube was stirred vigorously for 1 min. They were added 400 µl of phenol (saturated in water) / chloroform, returned to stir vigorously and phases were separated by centrifugation. The aqueous phase was passed to a clean eppendorf tube and adjusted to 2 M of ClLi. The mixture was kept at 4 ° C for 16 h and the precipitate It was collected by centrifugation. This precipitate (nucleic acids Total enriched in single strands) washed with 75% ethanol %, dried and resuspended in 100 µl of water. For the CVYV detection in plant nucleic acid extracts is  He proceeded as described below. One \ mul of the extract it was applied on a positively charged nylon membrane (Boehringer). The nucleic acids applied on the membrane are set by exposure to ultraviolet light on a device designed for this purpose (Ultraviolet Crosslinker; Amersham Life Sciences, UK). The membrane was then subjected to a process prehybridization by incubation at 65 ° C in buffer prehybridization (5xSSC, 0.1% N-Laurylsarcosine, 50% formamide, 0.02% SDS, 2% blocking agent [Boehringer]). To the after 2 h the prehybridization buffer was removed and the hybridization buffer (probe prepared as described previously diluted in prehybridization buffer in the proportion 1: 1000) in which the membrane was incubated at 65 ° C. The relationship between the membrane surface and the amount of hybridization buffer or prehybridization was 0.1 ml per cm2, with a minimum of 10 ml per membrane After 16 h of hybridization, the buffer is removed and stored frozen at -20 ° C for use in subsequent experiments The membrane underwent a process of washing that consisted of a 15 min incubation at temperature ambient in 2xSSC and 0.1% SDS and two 15 min incubations at 65 ° C in 0.1xSSC and 0.1% SDS. All three incubations were performed with gentle agitation and with a minimum wash buffer volume of 50 ml. The detection of the probe hybridized to the viral RNA present in The nucleic acid extracts were performed by Chemiluminescence using a Boehringer kit and following the manufacturer's instructions

CVYV detection by hybridization in imprints of sections of plant tissues

For the preparation of section imprints Plant tissue proceeded as follows. Stems, petioles and Major veins of plant leaves were sectioned with blade. To avoid contamination, each section was carried out with a sterile and distinct blade. The sections thus prepared are applied on a positively charged nylon membrane (Boehringer) until the exudated sap drops were absorbed. Once these applications were made, the membrane was kept at room temperature until dried. Acid fixation membrane nucleic, prehybridization, hybridization, washes and Detection of the probe hybridized to viral RNA was carried out in this case as described in the previous section.

The specificity and sensitivity of this method CVYV detection are illustrated below:

Specificity of the detection method

To determine the specificity of this detection method, the following experiment was designed. 3 seedlings of each of the following species were inoculated with the isolated CVYV-AlLM: melon, zucchini and cucumber, as well as 2 plants of each of the previous species with water only. After 3 weeks, the symptoms of infection were evident in the plants inoculated with virus while no symptoms appeared in plants inoculated with water. At this time, total nucleic acid extractions were made from these plants that were applied on membranes as described above. Also included imprints tissue sections of these plants in the membranes mentioned and applications extracts nucleic and imprints acids plants infected with virus the watermelon mosaic-II, the virus mosaic cucumber and virus the dwarfism and yellowing of the cucurbitaceae . These membranes were processed following the procedure object of the invention for the detection of CVYV. The results of this process showed that a signal appeared only in those samples from plants that were infected with CVYV, while in the healthy plants and in plants infected by viruses other than CVYV no signal appeared (Fig. 2). That is, the detection method is specific to CVYV.

Sensitivity of the detection method

The following experiment was carried out to determine the sensitivity threshold of this method. Viral RNA was prepared by in vitro transcription from plasmid pLMCVYV. To do this, plasmid pLMCVYV was linearized using the restriction enzyme PstI . For the in vitro transcription reaction, 10 µl of this linearized DNA was used at a concentration of 100 ng / µm to which 20 µl of 5-fold concentrated transcription buffer (Amersham Pharmacia Biotech), 10 µl of 100 mM DTT, 20 µl of a mixture of 2.5 mM rNTPs, 10 Units of RNase inhibitor (Amersham Pharmacia Biotech) and 50 Units of T7 transcriptase (Promega), all in a final volume of 100 µl. This mixture was kept at 37 ° C for 1 h 30 min. After this time, 10 units of RNase-free DNase (Boehringer) were added and incubation was continued for another 30 min. Then 4 µl of 0.5 M EDTA, 5 µL of 8 M ClLi and 300 µl of pure ethanol were added to the mixture, kept at -20 ° C for 3 h and the precipitated RNA was concentrated by centrifugation. The cleanliness and concentration of this RNA was determined by observing agarose gels stained with ethidium bromide on which electrophoresis of an RNA aliquot had been performed. RNA accurately quantified with respect to usual standards (Sambrook et al., 2001) was used as follows. From a concentrated solution at 0.6 µg / µl dilutions were made in water and in a healthy plant nucleic acid extract. The dilution factor was 1:10. One µl of each of these dilutions was applied on a membrane, as well as the corresponding diluents without viral RNA. This membrane was subjected to the CVYV detection process described above. The results of this experiment indicated that the minimum amount of viral RNA that can be detected with this method is 6 pg (Fig 3).

References

Square , IM, Janssen , D., Velasco , L., Ruiz , L. and Segundo E. ( 2001 ). First report of Cucumber vein yellowing virus in Spain. Plant Dis . 85: 336.

Mansour A. and Al-Musa A. (1993). Cucumber vein yellowing virus ; host range and virus vector relationships. J. Phytopathology 137: 73-78.

Matthews , REF ( 1991 ). Plant Virology Third Edition Academic Press, Inc. NY.

Narváez , G., Skander , BS, Ayllón , MA, Rubio , L., Guerri , J. and Moreno , P. ( 2000 ). A new procedure to differentiate citrus tristeza virus isolates by hybridization with digoxigenin-labeled cDNA probes. J. Virol. Methods 85: 83-92.

Lecoq , H., Desbiez , C., Delécolle , B., Cohen , S. and Mansour , A. ( 2000 ). Cytological and molecular evidence that the whitefly-transmitted Cucumber vein yellowing virus is a tentative member of the family Potyviridae. J. Gen. Virol . 81: 2289-2293.

Sambrook , J., Fritsch , EF and Maniatis , T. ( 1989 ). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Sela , I., Assouline , I., Tanne , E., Cohen , S and Marco , S. ( 1980 ). Isolation and characterization of a rod-shaped, whitefly-transmissible, DNA containing plant virus. Phytopathology 70: 226-228.

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Claims (7)

1. Procedure for the detection of the Cucumber vein yellowing virus (CVYV) in cucurbits characterized in that molecular hybridization techniques are used. 2. Method for the detection of the Cucumber vein yellowing virus (CVYV) in cucurbits according to claim 1, characterized in that it comprises the following steps:

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construction preparation genetics, a plasmid among others, that contains a fragment of the CVYV genome,

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preparation of an acid probe nucleic acids from the plasmid obtained in the previous step, Y

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probe hybridization with extracts of nucleic acids or with imprints of tissue sections of the plants to which the procedure applies.

3. Procedure for the detection of the Cucumber vein yellowing virus (CVYV) in cucurbits according to claim 2, characterized in that for the preparation of the genetic construct containing a fragment of the CVYV genome, two oligonucleotides are selected from the CVYV sequence. 4. Method for the detection of Cucumber vein yellowing virus (CVYV) in cucurbits according to claim 3, characterized in that the oligonucleotide used for the synthesis of the first cDNA chain is 5'-ACATTAGCGGCAAGTGTCTG-3 ' (SEQ ID NO 1) or any oligonucleotide complementary to at least 95% of its nucleotides to the CVYV sequence (SEQ ID NO 3). 5. Method for the detection of the Cucumber vein yellowing virus (CVYV) in cucurbits according to claim 3, characterized in that the oligonucleotide used in the construction of the second cDNA chain is 5'-GTCAGCCGTCAACTGTGGTGG-3 '(SEQ ID NO 2) or any oligonucleotide included in the CVYV nucleotide sequence (SEQ ID NO 3) and having a similarity of at least 95% with it. Method for the detection of the Cucumber vein yellowing virus (CVYV) in cucurbits according to claims 3-5, characterized in that the double cDNA chain obtained by using the oligonucleotides is subjected to ligation and subsequent insertion into a plasmid 7. Method for the detection of the Cucumber vein yellowing virus (CVYV) in cucurbits according to claim 6, characterized in that the resulting plasmid contains the sequence SEQ ID NO 3.