Classifications

• • 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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • 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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • 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
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to a new sequence specific for the phytoplasma causing Bois noir (BN) in vines, to a method for the diagnosis of BN in vines and parts thereof including rootstocks and to a kit for the detection of the phytoplasma agents of BN from plant samples.
• BN phytoplasma causing Bois noir
• Grapevine yellows a complex of diseases that were originally thought to be caused by viruses, are now known to have a phytoplasma aetiology.
• Bois noir (BN) whose symptoms are indistinguishable from those of FD, was also first reported from France, and then from the most important viticultural areas of Europe.
• GY syndrome Almost identical symptoms of the GY syndrome are caused by different phytoplasmas and appear on leaves, shoots and clusters of grapevine. Typical symptoms include discoloration and necrosis of leaf veins and leaf blades, downward curling of leaves, lack or incomplete lignifications of shoots, stunting and necrosis of shoots, abortion of inflorescences and shrivelling of berries. Those symptoms are related to callose deposition at the sieve plates and subsequent degeneration of the phloem. Although no resistant cultivars of Vitis vinifera or rootstocks are known so far, the various grape varieties differ considerably as far as symptom severity is concerned. It ranges from fast decline and death in highly susceptible cultivars to tolerant rootstocks as symptomless carriers of the pathogen.
• Phytoplasmas are microscopic plant pathogens, similar to bacteria, but much smaller than bacteria. They are phloem-limited, non-helical and wall-less prokaryotes (Firrao, G., et al, 2004.). Differently form viruses, phytoplasmas have their own metabolism, a highly reduced metabolism, and some of the molecules essential for their survival are acquired from the host. They represent a monophyletic clade within the class Mollicutes, which is currently divided into at least 15 subgroups on the basis of sequence analyses of various conserved genes. A new taxon, 'Candidatus Phytoplasma' has been established and various groups or subgroups have been described recently as 'Ca. Phytoplasma' species.
• GY have different phytoplasma species as causal agent, as well as different insect vectors, which are either leafhoppers or plant-hoppers (Homoptera: Auchenorrhyncha) that feed specifically or just occasionally on the vines. It is worth noting that two or more different phytoplasma species may infect simultaneously individual grapevines, thus causing mixed infections.
• phytoplasmas are non culturable micro-organisms and, in the case of GY, Koch's postulates have not yet been fulfilled, when phytoplasmas of a specific group or subgroup are found consistently associated with a specific grape disease, they are regarded as being its causal agents.
• the identification of phytoplasmas as the cause of GY was made possible over the last decades by molecular detection methods, which allow to distinguish each other the phytoplasma species involved in each single disease.
• phytoplasmas which are uncultivable and currently described under the provisional genus "Candidatus Phytoplasma,” is mainly based on 16S rRNA gene phylogeny, genomic diversity, and plant and insect host ranges.
• Aster yellows (16Srl-B), clover phyllody (16Srl-C) and elm yellows (16Sr-V) phytoplasmas were found sporadically in GY diseased grapevines in Italy, and infection of grapevine by aster yellows (16Srl-B) phytoplasma was reported from Tunisia. Infection of papaya and grapevine by "Ca. Phytoplasma australiense" was reported from Israel; however, confusion with stolbur phytoplasma which is also present in this region is possible.
• a survey in Italy revealed the presence of GY in 80% of the inspected vineyards. BN is widespread all over the country whereas FD is mainly restricted to the northern regions.
• phytoplasmas Among the most common ribosomal groups defined for phytoplasmas are: aster yellows (16Srl group), X-disease (16Srlll group), elm yellows (16SrV group) and solani also called potato stolbur phytoplasma (16SrXII group).
• BN is the most widespread GY in Europe and in the Mediterranean basin and it is caused by phytoplasmas belonging to stolbur group.
• the disease is present in almost all European vine areas as well as in Lebanon and Israel.
• the disease was firstly signalled in 1986 and the disease has assumed an endemic behaviour due to the principal vector of BN vector transmitting the disease that is Cixiidae planthopper Hyalesthes obsoletus.
• Cixiidae and Cicadellidae species are known or suspected vectors of stolbur phytoplasmas, too.
• Some additional species like Goniognathus guttulinervus in Sardinia and Reptalus panzeri in Hungary and Italy were found recently to carry solani (or stolbur) phytoplasma beside the already known species, although their ability to transmit the phytoplasma is still not proved.
• the BN vector does not feed only on vines and several alternative host plants of stolbur phytoplasmas play a vital role in the epidemiology of BN.
• Several herbaceous plants have been surveyed for the presence of 16Sr Xll-A phytoplasma infection and colonization by H. obsoletus and the host can hence transmit the disease from other plants to vines.
• Urtica dioica and Convolvulus arvensis that appear to be the major host plants of BN phytoplasma with high epidemic significance
• Vitex agnus-castus whose function as source of the phytoplasma is not yet clear
• Taraxum officinalis harbouring the phytoplasma and Ranunculus
• Solanum and Lavandula that also could serve as a source of inoculum.
• Vine appears to be a final host that does not function as source for the inoculation of the BN phytoplasmas.
• the multiplicity of hosts for this phytoplasma has practical effects that cannot be ignored, eradication of the diseased vines provides no help for the control or stop of the disease diffusion: it is therefore crucial to clearly identify a BN infection among other possible phytoplasma infections that can be confused with this disease in order to follow the best steps for the treatment of the vineyards, of the vineyards surrounding areas and or of the plant nurseries and garden centres.
• the symptoms caused by BN are practically the same as those of Flavescence doree, and the name refers to the blackening of non-lignified shoots in winter, which is a symptom of FD.
• FD phytoplasma While Flavescence doree (FD) phytoplasma is strongly epidemic and it is recognized by the European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest (EPPO/CABI, 2003), BN phytoplasma is not included in the quarantine pest list. Accordingly, the winegrower is not forced to eliminate the plants infected. However, it is important to identify the pathogen because the winegrowers can perform preventive strategies to limit the spread of disease.
• EPPO European and Mediterranean Plant Protection Organization
• BN detection has been developed; they include conventional PCR/RFLP methods based on analysis of ribosomal genes (Lee et al., 1994) and more recently real-time PCR assays (also RT PCR) have been developed (Galetto et al., 2005 ; Angelini et al., 2007 ; Hren et al., 2007; Margaria et al., 2009).
• PCR/RFLP analysis is relatively time consuming and requires several analytical steps involving samples cross-contamination risks. Also some RT PCR assays are available.
• the recently developed RT PCR assays are based on single nucleotide polymorphisms (SNPs) which identify BN phytoplasma. However, they target nucleotidic sequences (Angelini et al.2007; Hren et al., 2007) other than those of rpl22 and rps3 genes described in the present invention.
• SNPs single nucleotide polymorphisms
• the present description provides a new nucleotide sequence of SEQ ID NO 1 of coding for the gene rpl22 (ribosomal protein rpl22 gene) of phytoplasma "Ca. Phytoplasma solani” (Stolbur) causing BN, and SEQ ID NO 7 coding of the rps3 gene of phytoplasma "Ca. Phytoplasma solani” (Stolbur) causing BN. Both sequences are present in SEQ ID NO 1 , wherein rpl22 spans from nucleotide 1 to 390, and rps3 from nucleotide 374 to 1095. Both the rpl22 and rps3 genes code for ribosomal proteins.
• the rpL22 protein binds specifically to 23S rRNA during the early stages of 50S assembly and it makes contact with all 6 domains of the 23S rRNA in the assembled 50S subunit and ribosome; rpS3 forms a complex with S10 and S14 and it binds the lower part of the 30S subunit head and the mRNA in the complete ribosome to position it for translation.
• the newly provided sequences allow for the recognition of different phytoplasmas belonging to said subgroups and provide a new tool for a better understanding and recognition of these microorganisms.
• the present sequences i.e. SEQ ID NO 5, 7 and/or 1
• SEQ ID NO 5, 7 and/or 1 hence provide a useful tool for the specific detection of the presence of the BN related phytoplasma in a vine sample.
• the rpl22 and rps3 gene sequences specific for the BN related phytoplasma are depicted respectively in SEQ ID NO 5 and SEQ ID NO 7, and code for the ribosomal proteins rpL22 and rpS3 (respectively, SEQ ID NO 6 and SEQ ID NO 8) specific for the phytoplasma associated with BN that differs for single-nucleotide polymorphisms (SNPs) from other phytoplasmas such as "Ca. P. australiense,” “Ca. P. asteris", “Ca. P. vitis".
• SNPs single-nucleotide polymorphisms
• the present description also provides a sequence of SEQ ID NO 12 that corresponds to SEQ ID NO 1 wherein all SNPs nucleotides have been marked as "n" nucleotides and wherein n nucleotides can be any nucleotide including the one present in SEQ ID NO 1 , hence, a very specific embodiment of SEQ ID NO 12 is represented by SEQ ID NO 1 .
• sequences are workable as a diagnostic tool as they comprise various SNPs specific for BN.
• Each of said BN-specific SNP is identified in Table 1 where the BN specific nucleotide is indicated, as well as its position in SEQ ID NO 1 .
• nucleotides of Table 1 are the BN specific nucleotides whereas “nucleotides in the position indicated in Table 1 " may represent in an embodiment of the nucleotides of Table 1 but may also represent the "n” nucleotides of SEQ ID NO 12.
• the present invention hence provides a nucleotide sequence of SEQ ID NO
• the present invention also provides a nucleotide vector comprising a nucleotide sequence of SEQ ID NO 1 , 5, 7 or 12 or the sequence complementary to SEQ ID NO 1 , 5, 7 or 12 or fragments thereof, said fragments comprising one or more nucleotides in the position indicated in Table 1 or of their complementary nucleotides, and a method for the diagnosis of the vine disease Bois Noir comprising the step of
• Figure 1 represents an alignment of the rpl22 and rps3 genes of four phytoplasmas including "Ca. P. solani”, i.e, "Ca. P. australiense", “Ca. P. asteris” and “Ca. P. Vitis”.
• the alignment clearly demonstrates 66 SNPs in the positions reported below and in Table 1 below, each allowing the specific identification of phytoplasmas associated with BN.
• FIG. 1 Amplification plot of BN65 RT PCR assay. Specific detection of BN phytoplasma. The figure shows the amplification signal in positive control and in grapevine sample infected by BN. No amplification signal was detected in negative controls.
• SEQ ID NO 12 nucleotide sequence ofS10-spc operon comprising both rpl22 and rps3 nucleotide sequences
• the present description provides a new nucleotide sequence of SEQ ID NO 5 coding for the gene rpl22 (ribosomal protein rpl22 gene) of phytoplasma Ca. Solani (Stolbur) causing BN, and SEQ ID NO 7 coding of the rps3 gene of phytoplasma "Ca. P. solani "(Stolbur) causing BN.
• Both sequences are present in SEQ ID NO 1 , wherein rpl22 spans from nucleotide 1 to 390, and rps3 from nucleotide 374 to 1095.
• SEQ ID NO 1 comprises both rpl22 and rps3 nucleotide sequences and in particular, rpl22 spans from nucleotide 1 to 390, and rps3 from nucleotide 374 to 1095. Therefore, the position of each of the single SNPs above defined can be easily identified within the SEQ ID NO 5 (coding rpl22) and the SEQ ID NO 7
• the SNP defined as T in position 64 of SEQ ID NO 1 corresponds to the presence of nucleotide T in position 64 of SEQ ID NO 5;
• the SNP defined as C in position 252 of SEQ ID NO 1 corresponds to the presence of nucleotide C in position 252 of SEQ ID NO 5;
• the SNP defined as T in position 745 of SEQ ID NO 1 corresponds to the presence of T in position 371 of SEQ ID NO 7;
• the SNP defined as T in position 1010 of SEQ ID NO 1 corresponds to the presence of T in position 637 of SEQ ID NO 7.
• SNPs and the related nucleotide indicated in Table 1 is specific for the BN associated phytoplasma.
• SNPs and specific nucleotide indicated in table 1 above are also defined as “SNPs of Table 1 " and “nucleotides of Table 1 ", wherein the nucleotides of Table 1 are the ones specific for the BN associated phytoplasma per each identified SNP.
• SNPs disclosed in Table 1 are also numbered and can be identified in the present specification as “SNP 1 ", “2”, “3” and so on, and the SNP position as well as the nucleotide specific for the BN associated phytoplasma will be the corresponding ones in the same line of the Table.
• nucleotide sequence of SEQ ID NO 1 or the sequence complementary to SEQ ID NO 1 or fragments thereof comprising one or more nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ), or said fragments being suitable for the amplification of a fragment of SEQ ID NO 1 or of the sequence complementary to SEQ ID NO 1 comprising one or more of the nucleotides of Table 1 .
• SEQ ID NO 1 comprises both SEQ ID NO 5 and 7
• said sequences can be also defined (and are also defined in the present description) as "fragments of SEQ ID NO 1 comprising one or more of the nucleotides of Table 1 ".
• SNPs and specific nucleotides indicated above are also defined as SNPs of Table 1 and nucleotides of Table 1 , wherein the nucleotides of Table 1 are the ones specific for the BN associated phytoplasma per each identified SNP.
• the present description also provides an amino acid sequence of SEQ ID NO 6 coding for the rpl22 ribosomal protein and an amino acid sequence of SEQ ID NO 8 coding for the rps3 ribosomal protein, both of said proteins are specific for the BN phytoplasma belonging to the 16SrXII-A subgroup.
• a general sequence namely SEQ ID NO 12, wherein the nucleotides corresponding to the 66 SNPs allowing the BN phytoplasma identification listed in Table 1 are defined as "n" and can be any nucleotide.
• the corresponding amino acid general sequence can be derived automatically from the nucleotide sequence.
• the present description provides a nucleotide sequence of SEQ ID NO 1 or the sequence complementary to SEQ ID NO 1 or fragments thereof, said fragments comprising one or more of nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ), or said fragments being suitable for amplification of a fragment of SEQ ID NO 1 , or the sequence complementary to SEQ ID NO 1 comprising one or more of nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ) wherein said sequence or fragments thereof are specific for the plant phytoplasma strains associated with the vine disease BN.
• the sequence allowing the specific detection of the plant phytoplasma strains associated with the vine disease BN can hence be SEQ ID NO 1 or the sequence complementary to it, and the fragments can be fragments of SEQ ID NO 1 or fragments of the sequence complementary to it, provided that they comprise one or more of nucleotides of Table 1 (or of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 ), or that they are suitable for the amplification of a fragment comprising one or more of said nucleotides, as said particular nucleotides represents SNPs that renders SEQ ID NO 1 specific for the plant phytoplasma strains associated with the vine disease Bois Noir and allow, each alone or in combination with one or more of the others, a distinction of said phytoplasma from highly similar phytoplasmas that are not associated with the BN infection.
• the fragments should be fragments of a dimension suitable to be used for the detection of the phytoplasma strains associated with the vine disease BN with any common technique known for SNPs detection in the art. As already said, the fragments can also be represented by SEQ ID NO 5 and/or 7.
• the skilled person would know the suitable size for the fragments that could be from about 10, 20, 30, 40, 50, 60, 100, 150, 200, nucleotides to the full sequence of the gene rpl22 and/or rps3 for identification of the BN related phytoplasma.
• the fragments of the present description can be used as probes for PCR, as probes for microarray detection of SNPs, or can be the products of the techniques used for the SNPs identification.
• the fragments can be upstream or downstream fragments with respect to one or more of said nucleotides allowing amplification of a sequence comprising one or more of the nucleotides in the positions indicated in Table 1 or can be fragments comprising one or more of the aforementioned nucleotides.
• the identification of the nucleotide(s) of interest can be carried on either strand of the nucleotide sequence, hence, the fragments of the invention can be designed either to identify one or more of nucleotides in the positions indicated in Table 1 , and/or to identify one or more of their complementary nucleotides that are implicitly disclosed by the disclosure of SEQ ID NO 1 , 5, 7 and 12.
• the following description will clarify in more detail the fragments of the invention, all of them being characterised by the fact that they can be used for the identification of the aforementioned SNPs.
• SEQ ID NO 12 represents a generic sequence wherein the SNPs nucleotides of the alignment showed in figure 1 are indicated as "n”.
• nucleotide sequence of SEQ ID No 1 or fragments thereof are specific the phytoplasma associated with BN.
• SEQ ID NOs 1 , 5, and 7 represent each, one of the possible embodiments of SEQ ID NO 12 or fragments thereof, in particular the embodiment wherein the nucleotides in the positions indicated in Table 1 correspond to the nucleotides of Table 1 .
• sequences or fragments thereof herein described are applicable as diagnostic tools for the identification, in a grapevine sample, of the phytoplasma associated to the vine disease Bois noir.
• the invention also encompasses vectors such as cloning vectors or expression vectors comprising a nucleotide sequence of SEQ ID NO 12, 1 , 5 or 7 or fragments thereof, said fragments comprising one or more of nucleotides in the positions listed in Table 1.
• the vector of the invention can be used either as a positive or negative control in a diagnostic method for the detection of BN associated phytoplasma in a vine phloem sample depending on the nucleotides detected in the positions indicated in Table 1 (the exact nucleotides of Table 1 , will provide a positive control whether different nucleotides in the positions indicated in table 1 will provide a negative control) or it could also be used as research tool for the development of antibodies and/or for the study of possible therapies for curing the disease of the plant.
• the vector above can be represents, per se, a control for the method and the kit herein disclosed.
• the vector can also be used for cloning the genes of interest, for the production of the protein(s) coded, for the development of specific antibodies and in methods and studies for the development of therapies for the infected vines against BN.
• a further object of the invention is a method for the diagnosis of the vine disease Bois Noir comprising the step of
• the sample according to the present description can be any sample obtainable from a vine plant, provided that the sample comprises phloem of the plant due to the presence of the phytoplasmas being limited to this plant fluid.
• Phloem samples are also available from vine shoots or from the fruits or from the seeds or from the rootstock. Protocols for the collection of phloem-comprising samples are available to the skilled person without need of further details in the present description (Pasquini et al., 2001 ).
• the phloem obtainable by standard methods from the sample can be used as such, or an extraction of the total nucleic acids
• TAA can be carried out with standard methods (by way of example the method described in Angelini E, et al (2001 ) Flavescence doree in France and Italy: occurrence of closely related phytoplasma isolates and their near relationships to
• SNP ID 1 The identification of one or more nucleotides of the SNPs form SNP ID 1 to SNP ID 1 to
• 66 i.e. the nucleotides in the positions indicated in table 1
• the presence of one or more of the nucleotides of Table 1 in the sample analysed indicates the presence in said sample of the phytoplasma associated with the BN vine disease.
• the identification of step a) may comprise a first step of PCR amplification of one or more fragments comprising one or more of the SNPs from SNP ID 1 to 66 and a subsequent analysis of the amplified fragment either by assessing the mass of the amplified fragment(s) and hence defining the base pair present in the position of interest or by merely sequencing the fragment or by using the amplified fragment for other detection techniques as some of the techniques exemplified below.
• each a different amplicon size can be selected for the fragments in order to easy the detection.
• the identification step a) may comprise a first step wherein a RT PCR, Ligation, Allele Specific Hybridization, primer extension, invasive cleavage or sequencing reaction is carried out and a second step wherein the product obtained in the first step is detected by monitoring the light emitted by said product, by measuring the mass of said products, by monitoring the radioactivity emitted by said product or by sequencing said product.
• RT-PCR When RT-PCR is used, it is possible to apply a classic RT PCR protocol available in RT-PCR manuals and in laboratory protocols.
• the methods used to verify the identity of the amplicon(s) produced in RT PCR are sufficiently powerful to detect small variations between sequences. Variations in sequence, including SNPs have been successfully identified in RT PCR assays.
• One common approach to the detection of sequence variation is to compare melting curves. In general, the effect of base substitutions on the melting kinetics of PCR products is too small to be detected reliably; however, heteroduplexes of relatively long amplicons differing by a SNP can be distinguished from the homoduplexes on the basis of their melting curves.
• the melting curves of short fluorescent probes can be used to distinguish between amplicons. This method is sensitive to SNPs, which usually cause a shift in the melting peak of several degrees.
• the design of primers suitable for RT PCR is easily achievable with suitable programs available to the skilled person (a possible primer pair is depicted in SEQ ID NO 2 and 4) and the disclosure of SEQ ID NO 12, 1 , 5, and 7 and of all SNPs associated with BN phytoplasma, each of them reported in table 1 , is sufficient for the skilled person to readily design the RT PCR assay without use of inventive skill or cumbersome experimentation.
• SNPs nucleotides
• the skilled person will easily design primers and oligonucleotides for the amplification and detection of one o more nucleotides (SNPs) in the positions indicated in table 1 or for the amplification and detection of a fragment comprising one o more of said nucleotides or SNPs.
• RT PCR may involve the use of fluorescently labelled nucleic acid probes or primers, or DNA-binding fluorescent dyes such as SYBR® Green and others mentioned in the present description, to detect and quantify a PCR product at each cycle during the amplification. If desired, different fluorescent dies can be used for the different SNPs.
• the amplified product(s) or the probes can be labelled with a fluorophore following the manufacturer's instructions when melting curve of short fluorescent probes is analysed.
• RT-PCR approaches employ two different fluorescent reporters and rely on the energy transfer from one reporter (the energy donor) to a second reporter (the energy acceptor) when the reporters are in close proximity.
• the second reporter can be a quencher or a fluor.
• a quencher will absorb the energy from the first reporter and emit it as heat rather than light, leading to a decrease in the fluorescent signal.
• a fluor will absorb the energy and emit it at another wavelength through fluorescence resonance energy transfer (FRET, reviewed in 2), resulting in decreased fluorescence of the energy donor and increased fluorescence of the energy acceptor.
• FRET fluorescence resonance energy transfer
• a common alternative to the melting curve approach is to use hydrolysis (such as TaqMan) probes.
• Hydrolysis probes are labelled with a fluorescent dye at the 5'-end and a quencher at the 3'-end, and because the two reporters are in close proximity, the fluorescent signal is quenched.
• the probe hybridizes to PCR product synthesized in previous amplification cycles.
• the resulting probe: target hybrid is a substrate for the 5' ⁇ 3' exonuclease activity of Taq DNA polymerase, which degrades the annealed probe (3) and liberates the fluor. The fluor is freed from the effects of the quencher, and the fluorescence increases.
• the identification of the SNP of interest can be carried out by RT-PCR using suitable primer pairs and a suitable allele specific oligonucleotide probe (TaqMan ® probe), e.g. an MGB (Minor Groove Binding) probe.
• the allele-specific "TaqMan probe” may be designed based on the SNP information described above.
• the 5' end of TaqMan probe is labelled with fluorescence reporter dye R (e.g. FAM or VIC), and at the same time, the 3' end thereof is labelled with quencher Q (quenching substance).
• fluorescence reporter dye R e.g. FAM or VIC
• quencher Q quenching substance
• the TaqMan® MGB probes can be labelled with 6-carboxyfluorescein (FAM) at the 5' end and with a non-fluorescent quencher (NFQ) with minor groove binder (MGB) at the 3' end.
• FAM 6-carboxyfluorescein
• NFQ non-fluorescent quencher
• MGB minor groove binder
• MGB probes in the present invention disclosed a higher melting temperature (Tm) and increased specificity and were MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.
• RT-PCR reactions may be carried out on an ABI PRISM® 7300 Sequence Detection System (Applied Biosystems) in optical 96-well plates with optical adhesive covers (both Applied Biosystems) using the following cycling conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 61 ,5°C, which allowed running of all reactions on the same plate.
• RT-PCR can be performed in a final reaction volume of 25 ⁇ _ containing 5 ⁇ _ of sample DNA, 300 nm primers, 250 nm probe and 1 * TaqMan® Universal PCR Master Mix (Applied Biosystems), which includes ROX as a passive reference dye.
• Suitable primers and probes can be designed with various algorithms and programs available also on the web or the design thereof can be commissioned via commercially available services.
• suitable probes could be a forward primer of 21 nucleotides in length provided herein as SEQ ID NO 2, a TaqMan ® probe of 18 nucleotides in length provided herein as SEQ ID NO 3 labelled e.g. with 6-FAM at the 5' end, and a reverse primer of 24 nucleotides in length, provided herein as SEQ ID NO 4.
• the suitable annealing temperature is 60 °C.
• Assays using TaqMan ® are well known in the art and full protocols are available to the skilled person, in principle.
• the 5'-nuclease allelic discrimination assay, or TaqMan assay is a PCR-based assay for genotyping SNPs. The region flanking the SNP is amplified in the presence of two allele-specific fluorescent probes. The probes do not fluoresce in solution because of a quencher at the 3' end. The presence of two probes allows the detection of both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post-PCR processing.
• Allele Specific Hybridization also known as ASO (allele specific oligonucleotide hybridization).
• ASO allele specific oligonucleotide hybridization
• This protocol relies on distinguishing between two DNA molecules differing by one base (i. e. the SNP of interest) by hybridization.
• the TNA obtained by the phloem sample is amplified with suitable PCR primers designed in order to amplify a region of SEQ ID NO 12 comprising one or more of the nucleotides in the positions indicated in Table 1 (or of their complementary nucleotides in a sequence complementary to SEQ ID NO 12) or in other words of the SNPs of Table 1 , in a convenient embodiment, the PCR amplicons are fluorescence labelled.
• the fragments obtained by PCR are than applied to immobilized oligonucleotide fragments of SEQ ID No 1 comprising the SNP or the SNPs of interest.
• fluorescence intensity is measured for each SNP oligonucleotide.
• stringent hybridization and washing conditions will have to be used, where by stringent the skilled person will understand conditions that will allow only 100% matched hybridised sequences to remain in the double strand form.
• Typical means for regulating stringency of a hybridisation protocol are salt concentration (the highest the concentration the lowest the stringency) and/or formamide concentration (the highest the %, the highest the stringency) and/or temperature (the highest the temperature, the highest the stringency).
• Stringency conditions suitable for identification of SNPs by RT-PCR are known in the art and published on standard protocols for this reaction.
• This kind of identification can be carried out also in a solid phase, i.e. on a solid support (defined herein after) such as microtiter, microarray chip and the like, allowing the screening of a large number of samples.
• a solid support defined herein after
• microarrays will be used.
• the microarray slide is a very powerful diagnostic tool capable to identify, in a single experiment, the presence/absence of a high number of targets.
• the chips take advantage of an important DNA property, which is the complementary bases match (the T matches with the A and the G with the C) in its structure.
• the diagnostic technique consists of a luminous signal (emitted by a fluorophore at different wave lengths) in correspondence to the hybridization between the target fragment labelled with the fluorophore and the corresponding probe bound to the microarray slide. This binding, with subsequent light emission, shows that in the group of analysed probes a DNA fragment complementary to the probe that is "lighted" is present and, consequently, allows to know the identity of the target fragment.
• a PCR amplification of the target DNA is carried out in order to both increase the target DNA amount which will easy the detection of the signal, and in order to label the target DNA for the detection on the microarray.
• the labelling can be carried out with any labelling molecule commonly used for microarrays detection.
• the probe comprising a fragment of SEQ ID NO 1 , 5, and/or 7 wherein one or more nucleotides of table 1 , (and/or one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 1 , 5 and/or 7) can be placed per spot in order to increase the signal on the microarray when the phytoplasma associated to BN is present in the sample analysed and amplified.
• the region amplified will have to comprise at least one nucleotide corresponding to one or more of the nucleotides in the positions indicated in table 1 , i.e. the discriminating SNPs, in the amplicon(s).
• the region amplified hence can comprise one or more SNPs of interest and also more amplifications providing fragments comprising, each one of the SNPs of interest can be carried out.
• fragments comprising one or more of the nucleotides in the positions indicated in Table 1 (and/or one or more of their complementary nucleotides) are fragments wherein the nucleotides upstream and downstream said nucleotide(s) perfectly match with the nucleotides upstream and downstream the said nucleotide in SEQ ID NO 12 or in SEQ ID No 1 or in their complementary sequences when the complementary nucleotides are considered.
• perfectly match it is intended that a 100% alignment between SEQ ID NO 12 or SEQ ID No 1 or its complementary sequence and the fragment according to the present description is present.
• the design of the primers for the amplification can be carried out with any suitable program available to the skilled person (non limiting examples: Primer Premier, that can be used to design primers for single templates, alignments, degenerate primer design, restriction enzyme analysis, contig analysis and design of sequencing primers; AllelelD and Beacon Designer can design primers and oligonucleotide probes for complex detection assays such as multiplex assays, cross species primer design, species specific primer design and primer design to reduce the cost of experimentation; PrimerPlex is a software that can design ASPE (Allele specific Primer Extension) primers and capture probes for multiplex SNP genotyping using suspension array systems such as Luminex xMAP® and BioRad Bioplex, Primer Express ) or by commercially available services for oligo design.
• Primer Premier that can be used to design primers for single templates, alignments, degenerate primer design, restriction enzyme analysis, contig analysis and design of sequencing primers
• AllelelD and Beacon Designer can
• Suitable primers for the amplification of the fragment comprising the SNPs in position 610, 161 and 625 to be hybridised on the array are also the primers having SEQ ID NO 2 and SEQ ID NO 4 herein provided as exemplifying primers.
• hybridisation conditions for the allele specific hybridisation technique are available in the art, an example of suitable conditions can be 16 hours of hybridisation at 48°C (25% of formamide).
• microarray hybridisation could be carried out following a standard protocol as follows, wherein some step may be omitted, modified or added by the skilled person without the use of inventive skill:
• Microarray hybridization and wash conditions :
• Prehybridisation buffer 5x SSC, 0.1 % SDS and 1 % BSA. Heat to about 50°C while stirring; 2. Slides to be analyzed are placed in a staining jar; prehybridisation buffer is added, and incubation is carried out at about 48°C for 45- 60 min while stirring; 3. The slides are washed by dipping up and down approximately 10 times in two different staining jars of deionised water. Excess water is removed by shaking the slide rack up and down two times; 4. The slides are then dipped in an up and down motion approximately 10 times at room-temperature in isopropanol and spun dried. The slides are used immediately after prehybridisation (less than 1 hr) as hybridization efficiency decreases rapidly if the slides are allowed to dry for more than that time.
• 2X hybridization buffer 50% formamide, 10X SSC and 0.2% SDS. Incubate the solution until it reaches 48°C; 6. The labelled mixtures are re- suspended in 9 ⁇ water, and heated to 95°C for 3 min to denature, and are centrifuged at maximum angular velocity for 1 min.; 7. The following is added to each tube in order to block non-specific hybridization. Make a master-mix with the following ingredients for each tube:
• yeast tRNA (4mg/ml_) 2 ⁇
• the array is removed from the hybridization chamber with care taken not to disturb the coverslip; 13.
• the slide is placed in a rack for a staining dish containing 1 X SSC, 0.1 % SDS, and 0.1 mM DTT at about 48°C; 14.
• the coverslip is gently removed while the slide is in solution and agitated for 15 min.; 15.
• the slides are transferred to a staining dish containing 0.1 X SSC, 0.1 % SDS, and 0.1 mM DTT at about 48°C and agitated for 5 min.; 16. Repeat step 15 two more times; 17.
• the slides are transferred to a staining dish containing 0.1 X SSC and 0.1 mM DTT at room-temperature and agitated for 5 min.; 18. Repeat step 17 an additional time; 19.
• Slides are spun dried.
• Suitable labelling for the examples above, and for all the following examples where fluorescence is used for the detection can be carried out, e.g. with Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes or any other suitable commercial dye following the manufacturer's instructions.
• SBE single base extension
• the target region is amplified by PCR followed by a single base sequencing reaction using a primer that anneals one base inside of the polymorphic site.
• a primer that anneals one base inside of the polymorphic site.
• detection methods have been described. One can label the primer and apply the extension products to gel electrophoresis. Or the single base extension product can be broken down into smaller pieces and measured by Mass Spectrometry. The most popular detection method involves fluorescence labeled, dideoxynucleotide terminators that stop the chain extension.
• primer extension is a very robust allelic discrimination mechanism. It is highly flexible and requires the smallest number of primers/probes. Probe design and optimization of the assay are usually very straightforward.
• the skilled person starting from the sequence and the SNPs herein disclosed can easily and readily design suitable probes merely using commonly available software for probe design. Given the shortness of SEQ ID NO 5 and 1 and the one or maximum two SNPs to be identified, the design of the suitable probes will not be particularly difficult or toilsome and will not require the use of inventive skill, being the mere application of standard procedures or even commercially available services for probe design sufficient.
• primer extension approach There are numerous variations in the primer extension approach that are based on the ability of DNA polymerase to incorporate specific deoxyribonucleosides complementary to the sequence of the template DNA. However, they can be grouped into two categories: the first is a sequencing (allele- specific nucleotide incorporation) approach where the identity of the polymorphic base in the target DNA is determined; the second is an allele-specific PCR approach where the DNA polymerase is used to amplify the target DNA only if the PCR primers are perfectly complementary to the target DNA sequence.
• sequencing allele-specific nucleotide incorporation
• primer extension product analysis in homogeneous assays.
• a PCR product is produced.
• determining whether a PCR product is produced or not one can infer the allele found on the target DNA.
• Several approaches have been utilized to detect the formation of specific PCR products in homogeneous assays, e.g. based on melting curve analysis, or based on hybridization of target specific probes.
• a variation of this approach is the allele-specific primer extension.
• the PCR product containing the polymorphic site serves as template, and the 3' end of the primer extension probe consists of the allelic base. The primer is extended only if the 3' base complements the allele present in the target DNA. Monitoring the primer extension event, therefore, allows one to infer the allele(s) found in the DNA sample.
• SBE can also be easily carried out on microarrays using the well known SBE-TAG technique.
• Protocols suitable for carrying out this embodiment of the invention are described in manuals such as, by way of example, in "DNA microarrays: a molecular cloning manual” by David Bowtell, Joseph Sambrook, Protocol 6, pages 403-420, herein incorporated by reference.
• protocol 6 of the above mentioned manual all the teachings that are necessary to the skilled person for the design of this embodiment of the invention, from primer selection and design to buffers, is described in detail. The skilled person can hence easily carry out this SBE-tag embodiment of the invention starting from the teachings of the present description without use on inventive skill and without cumbersome preparations.
• Another protocol suitable for a SBE identification of the SNP of interest on microarrays is the Affymetrix tag array, the protocol being also available on manuals such as the manual mentioned herein above by Bowtell and Sambrook, described in detail in Protocol 7, pages 421 -428 herein incorporated by reference.
• a further suitable technique for the identification of the SNP(s) of interest is the ligation technique.
• the Allele Specific Oligonucleotide Ligation by designing oligonucleotides complementary to the target sequence, with the allele-specific base at its 3'-end or 5-'end, one can determine the genotype of the PCR amplified target sequence by determining whether an oligonucleotide complementary to the DNA sequencing adjoining the polymorphic site is ligated to the allele-specific oligonucleotide or not.
• This assay relies on the fact that DNA ligase is an enzyme that is highly specific in repairing nicks in the DNA molecule. When two adjacent oligonucleotides are annealed to a DNA template, they are ligated together only if the oligonucleotides perfectly match the template at the junction.
• Allele-specific oligonucleotides can, therefore, interrogate the nature of the base at the polymorphic site.
• Ligation has the highest level of specificity and it is the easiest to optimize among all allelic discrimination mechanisms, but it is the slowest reaction and requires the largest number of modified probes.
• ligation as a mechanism has the potential of genotyping without prior target amplification by PCR. This can be accomplished either by the ligation chain reaction (LCR) or by the use of ligation (padlock) probes that are first circularized by DNA ligase followed by rolling circle signal amplification.
• the ligation technique is applicable on microarrays, in this case, the technique is called more specifically Ligation Detection Reaction - Universal Array (LDR-UA) and allows the detection of Single Nucleotide Polymorphisms (SNPs) on DNA molecules.
• LDR-UA Ligation Detection Reaction - Universal Array
• SNPs Single Nucleotide Polymorphisms
• the LDR-UA technique takes advantage of two different probes, called Common Probe (CP) and Discriminating Probe (DP), which are designed to anneal juxtaposed on target single strand DNA. DP anneals on the DNA at the 5' end of CP.
• the two probes can be ligated by a thermo-stable ligase such as the Pfu Ligase. If the complementarity between the 3' end of the DP and the target DNA is not perfect, the ligation reaction is compromised. For this reason the last base at the 3' end of the DP is also called discriminating base.
• Each DP must be labelled with a distinct fluorophore at its 5' end.
• the CP must be phosphorylated at its 5' end and must be extended at its 3' end with a "cZIP Code” sequence, which is the complementary and inverse of the "Zip Code” sequence spotted on the Universal Array. Every CP corresponds to a different "ZIP Code”.
• the ligation product is hybridized to the UA.
• Each UA spot is composed of different "Zip Code” DNA sequences that capture the corresponding "cZIP Code” sequences at the CP 3' end.
• the CP-DP ligation event positions the fluorophore at the 5' end of the DP on the corresponding UA spot that is visualized by the subsequent scanning of the UA. The signal from a spot therefore indicates the perfect match between the DP and the target DNA.
• the identification of the SNP(s) in the positions indicated in Table 1 can be carried out by the invasive cleavage technique as described, e.g. by Wilkins Stevens P. et al 2001 .
• the invasive cleavage assay is a probe cycling, signal amplification reaction used for detection of single nucleotide polymorphisms (SNPs).
• SNPs single nucleotide polymorphisms
• the reaction requires two synthetic oligonucleotides, called the 'upstream oligonucleotide' and 'probe', that anneal to the target sequence with an overlap of 1 nt. This creates a bifurcated overlapping complex that resembles a structure generated during strand displacement DNA synthesis.
• Structure-specific 5'-nucleases whose primary cell function is believed to be processing of Okazaki fragments, cleave the bifurcated substrate at the site of the overlap, releasing the 5'-arm and one base paired nucleotide of the probe.
• the cleaved 5'-arm serves as a signal indicating the presence of target in an analyzed sample.
• Tm probe melting temperature
• multiple cleavage events can be achieved for each target.
• an invasive signal amplification reaction generates 30-50 cleaved probes/target/min, resulting in 103-104-fold signal amplification in a 1-3 h reaction.
• the unique ability of 5'-nucleases to specifically cleave the overlapping substrate can be utilized for detection of single base mutations.
• the upstream oligonucleotide and probe are designed to create overlap at this nucleotide, ensuring efficient cleavage of the probe.
• a substitution at this nucleotide position eliminates the overlap and dramatically reduces the cleavage rate, resulting in mutation discrimination of at least 300:1.
• Such discriminatory power makes the invasive cleavage assay an excellent tool for identification of SNPs.
• Signal detection can be carried out by electrophoresis, microplate (microtiter) enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry methods, and fluorescence resonance energy transfer (FRET) methodology.
• ELISA enzyme-linked immunosorbent assay
• MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
• FRET fluorescence resonance energy transfer
• the invasive cleavage assay is adaptable to a solid phase format presenting the possibility of analyzing multiple SNPs or multiple samples for a single SNP in parallel.
• SNP detection using the invasive cleavage reaction can be performed in 96-well microplates with nanogram amounts of DNA per SNP.
• the SNP can be identified by the SniPer method, which allows discriminating alleles by examining the presence or absence of amplification by RCA (rolling circle amplification).
• the DNA to be used as a template is linearized.
• a probe is hybridized to this linearized DNA.
• the genomic DNA can be converted into a circular DNA through ligation reaction.
• RCA of the circular DNA proceeds.
• the ends of the probe do not match with the genomic DNA, the DNA is not ligated to become a circular DNA. Thus, RCA reaction does not proceed.
• a single-stranded probe which anneals with the genomic DNA and is circularizable is designed.
• This single- stranded probe is called a padlock probe.
• the sequences of the two ends of this padlock probe are designed so that they correspond to the SNP to be detected. Then, this padlock probe and the genomic DNA are mixed for ligation. If the two ends of the padlock probe and the SNP site of the genomic DNA are complementary to each other, the two ends of the padlock probe are joined by ligation, yielding a circular probe. If the two ends of the padlock probe and the SNP site of the genomic DNA are not complementary to each other, the probe does not become circular.
• padlock probes which are complementary to the SNP to be detected become circular and are amplified by DNA polymerase. By detecting the presence or absence of this amplification, SNP may be detected.
• synthetic oligonucleotides which have a fluorescent dye and a quencher at their respective ends and also have a hairpin structure are used.
• a further technique applicable to the diagnostic method herein disclosed is the amplification of a fragment comprising one or more of the nucleotides in the positions indicated in Table 1 and sequencing of the same with direct identification of one or more said nucleotides (and/or of their complementary nucleotides).
• the sequencing can be readily and easily performed with automated sequencers.
• the amplification primers of SEQ ID NO 2 and 4 are suitable also for this technique. It is evident that any primer pair suitable for the specific amplification of all or part of SEQ ID NO 12 or 1 provided that one or more of nucleotides in the positions indicated in Table 1 are comprised in the amplicon obtained (and/or one or more of the complementary nucleotides), or any probe overlapping with one or more of the nucleotides in the positions indicated in Table 1 (and/or one or more of the complementary nucleotides) can be used for carrying out the method of the invention, and that the invention is not limited to primers of SEQ ID NO 2-4.
• a control could be carried out by detecting also the allele carrying the non BN specific allele. It is evident that in a kit for carrying out the diagnostic method herein described suitable probes can be provided also for the detection of the non BN specific allele.
• the diagnostic method herein disclosed can be carried out on solid supports.
• Suitable solid supports can be a latex bead, a glass slide, a silicon chip, or the walls of a microtiter well.
• marker specific oligonucleotides are placed on the solid support, and the allelic discrimination reaction is done on the support whereas in other cases, generic oligonucleotides are placed on the solid support, and they are used to capture complementary sequence tags conjugated to marker specific probes.
• the oligonucleotide arrays act as a collection of reactors where the target DNA molecules find their counterparts, and the allelic discrimination step for numerous markers proceeds in parallel.
• the arrayed oligonucleotides are used to sort the products of the allelic discrimination reactions (also done in parallel) performed in homogeneous solution.
• allelic discrimination reactions also done in parallel
• the identity of an oligonucleotide on a latex bead or at a particular location on the microarray (on a glass slide or silicon chip) is known, and the genotypes are inferred by determining which immobilized oligonucleotide is associated with a positive signal.
• the clear advantage of performing genotyping reactions on solid supports is that many markers or, in the present case many samples, can be interrogated at the same time. Besides saving time and reagents, performing numerous reactions in parallel also decreases the probability of sample/result mix-ups.
• the detection of the product obtained by the first step of the identification step a), e.g. the product obtained by the aforementioned techniques, can be performed by monitoring the light emission, by measuring of the mass, by monitoring of the radioactivity or by sequencing of the said product.
• said monitoring of the light emission can be carried out by monitoring fluorescence, luminescence, time resolved fluorescence, fluorescence resonance energy transfer, or fluorescence polarisation
• said measuring of the mass can be carried out by mass spectrometry and said monitoring of the radioactivity can be carried out with radioactivity sensitive tools.
• Monitoring light emission is the most widely used detection modality in genotyping, this can be done by measuring or detecting Luminescence, fluorescence, time resolved fluorescence, fluorescence resonance energy transfer (FRET), and fluorescence polarization (FP).
• FRET fluorescence resonance energy transfer
• FP fluorescence polarization
• Luminescence is emitted in an ATP-dependent luciferase reaction.
• ATP production is coupled with a primer extension reaction
• luminescence is observed every time a deoxyribonucleoside is added in the primer extension reaction.
• Luminescence can be measured with suitable commercial analysers following the manufacturer's instructions (e.g. Applied Biosystems 1700 Chemiluminescent Microarray Analyzer).
• Fluorescence can be measured using commercially available fluorescence sensitive imaging devices or measuring devices known in the art and labelled probes or amplified products according to the selected technology for carrying out the method herein described.
• Suitable fluorophores for labelling the nucleic acids of interest can be selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes.
• Detection can be carried out following the manufactures instructions.
• the skilled person is well aware that all techniques indicated above can all be carried out with fluorophores following conventional protocols.
• Detection by Time-resolved fluorescence can be made with dyes having a long half life (such as Lanthanides), the fluorescence reading is done sufficiently long after excitation, such that autofluorescence (which has a very short half-life) is not observed.
• dyes having a long half life such as Lanthanides
• autofluorescence which has a very short half-life
• lanthanides are inorganic compounds that cannot be used to label nucleic acids directly an organic chelator conjugated to the probe must be used to bind the lanthanides in the reaction. Protocols are available for this kind of detection.
• Fluorescence resonance energy transfer occurs when two conditions are met. First, the emission spectrum of the fluorescent donor dye must overlap with the excitation wavelength of the acceptor dye. Second, the two dyes must be in close proximity to each other because energy transfer drops off quickly with distance. The proximity requirement is what makes FRET a good detection method for a number of allelic discrimination mechanisms. Basically, any reaction that brings together or separates two dyes can use FRET as its detection method. FRET detection has, therefore, been used in primer extension and ligation reactions where the two labels are brought into close proximity to each other.
• FP can be used in several SNPs detection techniques, commercial systems such as the Perkin Elmer AcycloPrimeTM-FP SNP Detection System are available to the skilled person.
• MS mass spectrometry
• a further method requires the use of radiolabels instead of fluorescent labels and in this case radioactivity of the sample is measured in standard ways well known in the art.
• Another embodiment of the present invention is a diagnostic kit for the diagnosis of the vine disease Bois noir comprising reagents for the identification of one or more of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 12 and/or of one or more of their complementary nucleotides in the sequence complementary to SEQ ID NO 12 in a plant phloem.
• the kit of the present invention comprises any and all components enzymes or components necessary (suitable) for an intended assay.
• components include, but are not limited to, labelled and/or non labelled oligonucleotides, polymerases (e.g. Taq polymerase), buffers (e.g. Tris buffer), dNTPs labelled or non labelled, control reagents (e.g. tissue samples, target oligonucleotides for positive and negative controls, etc.), labelling and/or detection reagents (fluorescent dyes such as VIC, FAM), solid supports, manual, illustrative diagrams and/or product information, inhibitors, and packing environment adjusting agents (e.g. ice, desiccating agents).
• polymerases e.g. Taq polymerase
• buffers e.g. Tris buffer
• control reagents e.g. tissue samples, target oligonucleotides for positive
• the kit of the present invention may be a partial kit which comprises only a part of the necessary components. In this case, users may provide the remaining components.
• the kit of the present invention may comprise two or more separate containers, each containing a part of the components to be used.
• the kit may comprise a first container containing an enzyme and a second container containing an oligonucleotide.
• Specific examples of the enzyme include also a structure-specific cleaving enzyme, ligases or other enzymes for use in the techniques for identification and detection described above contained in an appropriate storage buffer or a container.
• oligonucleotide examples include nucleotides for the RT-PCR, nucleotides for the extension technique described above, nucleotides the ligation technique described above, oligonucleotides for the LDR-UA technique described above, oligonucleotides for the invader technique described above, probe oligonucleotides for the hybridisation technique described above, target oligonucleotides for use as control, and the like.
• the oligonucleotides can be labelled according to the technique selected.
• reaction components may be provided in such a manner that they are pre-divided into portions of a specific amount. Selected reaction components may also be mixed and divided into portions of a specific amount. It is preferred that reaction components should be pre-divided into portions and contained in a reactor. Specific examples of the reactor include, but are not limited to, reaction tubes or wells, or microtiter plates. It is especially preferable that the pre- divided reaction component should be kept dry in a reactor by means of, for example, dehydration or freeze drying.
• the kit of the invention may further comprise solid supports wherein oligonucleotides for the identification of one or more of the nucleotides the positions indicated in Table 1 in SEQ ID NO 12 and/or of one or more of their complementary nucleotides in the sequence complementary to SEQ ID N012 are anchored in known positions on said support in one or more copy per position.
• the anchorage of the oligonucleotides/probes as described above in the section related to the techniques suitable for carrying out the method will be anchored to the solid support by standard techniques selected depending on the support chosen.
• the kit of the invention may comprise labelled probes corresponding and/or complementary to a region of SEQ ID NO 12 upstream and/or downstream of one or more of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 12 and/or overlapping a region comprising one or more of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 1.
• Upstream and downstream oligonucleotides may be directly adjacent to one of the nucleotides in the positions indicated in Table 1 of SEQ ID NO 1 or to its complementary nucleotide in the sequence complementary to SEQ ID NO 1 e.g.
• kits for identification by ligation or by other techniques as exemplified above requiring probes directly adjacent to the SNP; or may be non directly adjacent to the SNP nucleotide when the kits are for identification with techniques as exemplified above that to not require probes directly adjacent to the SNP.
• probes and oligonucleotides of the invention fall within the definition of fragments of SEQ ID NO 1 or of its complementary sequence and will be of a dimension comprised between about 8 to about 100 nucleotides, normally of a dimension comprised between about 15 to about 50 nucleotides.
• fragments and probes do not comprise one or more the SNPs of interest but allow their identification (e.g. when the fragments are amplification fragments) they also fall within the definition of fragments of SEQ ID NO 1 or 12 or of their complementary sequence and will be of a dimension comprised between about 8 to about 100 nucleotides, normally of a dimension comprised between about 15 to about 50 nucleotides.
• the fragments include also fragments that are larger than the oligonucleotides and probes, could be represented e.g. by amplicons and can span from the dimensions of the oligonucleotides and probes indicated above, to a length equal to the length of SEQ ID NO 1 or 12 minus 1 nucleotide.
• the probes and oligonucleotides of the kit may be labelled with a fluorophore, e.g. with a fluorophore selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes.
• a fluorophore selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein, 6-Fam, Hex, Tet, Tamra, Joe, Rox, IRDyeTM700, IRDyeTM800, Dyomics Dyes, Atto Dyes.
• the kit may also comprise the vector of the invention either as a the positive or negative control as described above, or may comprise both a positive and a negative control i.e. the positive control will comprise BN associated a vector encoding a sequence comprising phytoplasma's specific SNP and/or SNPs disclosed in the present description (i.e. from SEQ ID NO 1 , 5 or 7) and the negative control will comprise at the disclosed SNP or SNPs site/s a sequence of SEQ ID NO 12 wherein said sequence is not SEQ ID NO 1.
• monoclonal or polyclonal antibodies specifically recognising SEQ ID No 6 and/or 8 can be prepared according to standard techniques and even purchased by companies specialised in antibody preparation starting from SEQ ID No 6 and/or 8.
• the antibody can be produced by standard techniques
• the antibody can be produced according to any known standard technique such as described also in the monoclonal and polyclonal production related chapters in the manual "Basic Methods in Antibody Production and Characterization" edited by G.C. Howard and D.R.
• BN associated phytoplasma can be carried out by immunological assays on vine phloem samples where cell lysis has been carried out to expose rpl22-rps3 encoded ribosomal proteins of SEQ ID No 6 or 8 expressed inside the infected cell.
• the present invention hence also encompasses a method for the detection of BN associated phytoplasma in a vine sample wherein a primary antibody specifically recognising SEQ ID NO 6 and/or 8 and a labelled secondary antibody specifically recognising the primary antibody are used in an immunodetection assay on a vine phloem sample wherein cell lysis has been carried out.
• TAA Total nucleic acids
• strains EY1 ('Ca. Phytoplasma ulmi', subgroup 16SrV-A), STOL (stolbur group, subgroup 16SrXII-A), and AY1
• DNA sequencing was performed in an ABI PRISM 377 automated DNA sequenze (Applied Biosystems).
• the nucleotide sequence data were assembled by employing the Contig Assembling program of the sequence analysis software BIOEDIT, version 7.0.0 (http://www.mbio.ncsu.edu/Bioedit/bioedit.html).
• the size of the nucleotide fragment was 1 122bp and included rpl22 gene sequence and partial sequence of rps3 gene.
• GenBank in order to identify sequence variation that would allow identifying regions suitable for design of diagnostic assays.
• Taqman PCR amplification was done in a 25 ⁇ final volume including 5 ⁇ of purified DNA as template.
• the PCR Master Mix included BN-701 (TaqMan® forward primer), BN-65 (TaqMan® probe) and BN-771 (TaqMan® reverse primer) and amplification conditions as follows: 94 °C for 10 min, 94 °C for 30 s, 60 °C for 1 min, and amplification of 40 cycles.
• the optimal condition for primers and probe concentration was respectively 900nM (both primers) and 250 nM (probe).
• Fig. 2 is showed an amplification plot example.
• Negative control AY negative negative negative 16Srl-A FD negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative
• the amplified genomic DNA is labelled with the "BioPrime® Total Genomic Labeling System” kit (INVITROGEN - 18097-012) following the manufacturer's instructions.
• the labelled sample is precipitated by Spin - Vacuum Savant and is subsequently re suspended in the hybridisation solution.
• Probes comprising one or more the SNPs region of interest have been blocked on the slide for the detection of the phytoplasma SNP(s) associated with BN disclosed in the present description, in 16 copies per probe.
• the activation of the slide shall be carried out (glass surface chemistry: EPOXY Surface Coating Slides; spotting buffer: Scott-Nexterion spotting buffer; probe's concentration: 30 ⁇ ), by the use of a blocking solution (10x Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulfate (SDS), 0.066 Sodium Tetrahydridoborate (NaBH 4 ), H 2 0 up to 50ml).
• the slide is treated with the blocking solution for 20 minutes to 42°C. Washings are carried out: twice (Sodium Saline Citrate 1 x for 5 minutes at room temperature), twice (Sodium Saline Citrate 0.1 x for 5 minutes at room temperature).
• the solution must be filtered by 0.2 ⁇ filters.
• the slide area comprising the probes is delimited with a "frame” (Gene Frame 21x22mm and cover slips - AB1043 CELBIO). 1 10 ⁇ of prehybridisation solution are placed within this area and the slide is covered with the cover slip. The slide with the prehybridisation solution is incubated at 42°C for 2 hours.
• a solution consisting of: 5X Sodium Saline Citrate (SSC), 0.1 % Sodium Dodecyl Sulfate, 25% Formamide, 200 ⁇ g Salmon Sperm DNA, H 2 0 up to 2ml is prepared.
• the solution must be filtered by 0.2 ⁇ filters and preheated at 42°C.
• the sample of the amplified and labelled DNA is re suspended in about 1 10 ⁇ of hybridisation solution.
• the DNA sample, resuspended in hybridisation solution is denatured at 95°C.
• the hybridisation solution is placed in the centre of the "frame” that defines the area comprising the target probes, and the cover slip is placed on this area. The slide is hence incubated to 42°C for 16 hours.
• Post-hybridisation washing :
• the slide has been dried by centrifugation to 800 rpm for 5 minutes.
• MLO mycoplasmalike organism

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