Patent Application
20090105238
The present invention relates to compounds and the relative use thereof for the control of phytopathogens.
Amphoteric surface-active agents, such as alkyl betaine, alkylamide alkyl betaine, hydroxysulfobetaine, are compounds which are known for their foaming, viscosizing, antistatic, softening properties, and thanks to their excellent affinity with other types of surface-active agents and intrinsic low irritating capacity with respect to the skin and eyes, are widely used in detergents and cosmetics.
It is also known that the above amphoteric surface-active agents can be used as components in formulations of agro-drugs as described, for example, in WO-A-97/47196 and EP-B-0597488 and in numerous other patents.
EP-A2-1542023, moreover, claims the use of amphoteric surface-active agents as “bioactivators” of agro-drugs already on the market, in suitable agronomic applications. In particular, their mixing with a herbicidal compound, such as for example, Glyphosate, improves its biological activity. It should be pointed out that the effect of “bioactivators” is exerted in an increased absorbability of the agrochemical active principle (herbicide, fungicide, insecticide, acaricide . . . ) inside the tissues of the plant or surface of the pathogen, or in an increased availability of the agrochemical active principle for the organisms of interest.
The compositions described in EP-A2-1542023 therefore allow a reduction in the applied concentrations of the active principles thus added.
In EP-A2-1542023, the amphoteric surface-active agents consequently merely act as a carrier of the active principles with which they are simply mixed, according to the logical role of a formulation component. A biological activity of the above surface-active agents is expressly excluded.
In the agronomical field, moreover, it is known that glycine betaine, when administered to fruit plants, contributes towards controlling abiotic and nutritional growth stress, reducing imperfections in the fruit peel and the tendency of the peel to break when ripening, as described in EP-A-0806897, acting as an osmolyte regulator.
The Applicant has now surprisingly found various amphoteric compounds which have a surprising activity in the agronomical field, as fungicidal and bactericidal products and which allow a prolonged protective action to be obtained on plants with respect to phytopathogen fungi and bacteria.
An object of the present invention is therefore an amphoteric compound characterized by a zwitterionic structure of the betainic type having general formula (I),
wherein:
The Applicant has also found that the compounds having general formula (I), in addition to having a direct fungicidal and bactericidal action, are capable of stimulating the natural defense systems of plants and inducing resistance in the plant itself; this method for controlling diseases and mitigating abiotic stress (temperature, salinity, drought, etc.) and biotic stress, is becoming of increasing interest, as it is based on the amplification of a natural process already present in the plant by the application of these compounds.
The Applicant has also surprisingly found that these compounds having general formula (I) represent an optimum form for controlling phytopathogens also in genetically modified vegetable varieties for amplifying the original natural defense.
A further object of the present invention therefore relates to the use of amphoteric compounds having a zwitterionic structure of the betainic type having general formula (I):
wherein:
Furthermore, an object of the present invention relates to the use of amphoteric compounds having a zwitterionic structure of the betainic type having general formula (I) for the stimulation of the natural defense systems of plants from abiotic and biotic stress and the induction of resistance in the plant itself.
In particular, the use of the compounds having general formula (I) for the control of phytopathogen fungi is curative and/or preventive.
Furthermore, said use for the control of phytopathogen is also effected in genetically modified vegetable varieties.
A further object of the present invention also relates to the use of said compounds having general formula (I) for the control of fungal diseases also on non-living substrates, such as for example, plastic materials, metals, textile fibres, glass, wood, paper, foams, bricks, etc. Said compounds can be applied to the surface of the substrate by means of methods well known in the art, such as for example, spraying, painting, immersion, impregnation, etc., at application doses depending on the kind of material and conditions to which the substrate is subjected.
A C1-C26 alkyl group refers to a linear or branched C1-C26 alkyl group, optionally substituted by one or more substituents the same or different.
Examples of this group are: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, capryl, lauryl, stearyl, eicosyl, hexacosyl.
A C1-C26 haloalkyl group refers to a linear or branched alkyl group, substituted by one or more halogen atoms, the same or different.
Examples of this group are: fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, 2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl, perfluoro-octanyl, perfluorododecyl.
A C1-C26 alkoxyl group refers to a C1-C26 alkoxyl group, wherein the aliphatic portion is a C1-C26 alkyl, as previously defined.
Examples of this group are: methoxyl, ethoxyl, isopropoxyl, cyclopropylmethoxyl, lauryloxyl.
A C1-C26 thioalkyl group refers to a C1-C26 thioalkyl group, wherein the aliphatic portion is a C1-C26 alkyl, as previously defined.
Examples of this group are: thiomethyl, thioethyl, thiolauryl, thiocapryl.
A C2-C26 alkenyl group refers to a linear or branched C2-C26 alkenyl group, optionally substituted by one or more substituents the same or different.
Examples of this group are: ethenyl, propenyl, butenyl, 1-decenyl, 8-heptadecenyl, 8,11,14-heptadecatrienyl, 8,11-heptadecadienyl.
A C2-C26 alkinyl group refers to a linear or branched C2-C26 alkinyl group, optionally substituted by one or more substituents the same or different.
Examples of this group are: ethinyl, propargyl, 1-dodecinyl, 1-octadecinyl.
A C3-C30 cyclo-alkyl group optionally condensed refers to a cyclo-alkyl group whose ring consists of 3-30 carbon atoms, optionally substituted by one or more substituents the same or different.
Examples of this group are: cyclopropyl, 2,2-dichlorocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decaline, abietyl.
A condensed C17 cyclo-alkyl group of the steroid type refers to a steroid group consisting of 17 carbon atoms, optionally substituted by one or more substituents the same or different.
Examples of this group are: cholanyl, or chenodeoxycholanyl, or ursodeoxycholanyl, or deoxycholanyl, or iodeoxycholanyl, or lithocholanyl.
A C3-C30 cyclo-alkoxyl group refers to a C3-C30 cyclo-alkoxyl group wherein the aliphatic portion is a C3-C30 cyclo-alkyl group as previously defined.
Examples of this group are: cyclopentoxy, cyclohexyloxy, cholesteryl.
A C1-C26 alkylamine or a C2-C26 dialkylamine group refers to an alkylamine or dialkylamine group wherein the aliphatic portion is respectively a C1-C26 or two C1-C13 alkyl groups as previously defined.
Examples of this group are: methylamine, dimethylamine, ethylamine, isopropylamine, dibutylamine, dioctylamine, hexadecylamine, dodecylamine.
An aryl group refers to an carbocyclic aromatic group optionally substituted by one or more groups the same or different.
Examples of this group are: phenyl, naphthyl, phenanthryl.
A hetero-aryl group refers to a penta- or hexaatomic heterocyclic aromatic group also benzocondensed or heterobicyclic, containing from 1 to 4 hetero-atoms selected from nitrogen, oxygen, sulfur, optionally substituted by one or more groups the same or different.
Examples of hetero-aryl groups are: pyridine, pyrimidine, pyridazine, pyrazine, triazine, tetrazine, quinoline, quinoxaline, quinazoline, furan, thiophene, pyrol, oxazole, thiazole, isoxazole, isothiazole, oxadiazole, thiadiazole, pyrazole, imidazole, triazole, tetrazole, indole, benzofuran, benzothiophene, benzoxazole, benzothiazole, benzoxadiazole, benzothiadiazole, benzopyrazole, benzimidazole, benzotriazole, triazolepyridine, triazolepyrimidine, thiazoltrizole, cumarin.
A heterocyclic group refers to a saturated or unsaturated ring with three to twelve terms, containing at least a heteroatom selected from nitrogen, oxygen, sulfur, optionally condensed with another aromatic or non-aromatic ring.
Examples of heterocyclic rings are: pyrrolidine, piperidine, dihydropyridine, piperazine, 2,6-diketopiperazine, 2-ketoazetidine, morpholine, thiazine, indoline.
A linear or cyclic C6-C12 group of the saccharide type refers to a carbohydrate group in open or closed form.
Examples of this group are: gluconyl, glucopyranosyl, β-D-fructofuranosyl-α-D-glucopyranosyl, 4-O-β-D-galactopyranosyl-D-glucosyl.
Optionally substituted means, in all parts of the patent, one or more substituents, the same or different, selected from the following groups: halogen atoms; C1-C6 alkyls, C1-C6 alkoxyls and C1-C6 alkylthio, in turn optionally substituted by halogen atoms; C1-C6 alkylcarbonyls and C1-C6 alkoxycarbonyls, optionally halogenated; aminocarbonyls, C1-C6 alkylaminocarbonyls, C2-C12 dialkylaminocarbonyls, optionally halogenated; carboxyl; C1-C6 alkylcarbonyloxy optionally halogenated; cyano; nitro; formyl; hydroxyl; amino; aryl and hetero-aryl optionally substituted.
Examples of compounds having general formula (I) which are interesting for their activity are:
The compounds having formula (I), when R1 has the meanings defined above with the exclusion of a C1-C26 alkoxyl group, or a C1-C26 alkylthio group, or a C3-C30 cyclo-alkoxyl group, or a C1-C26 alkylamine group, or a C2-C26 dialkylamine group, can be easily obtained according to reaction scheme A for n different from 0 and according to reaction scheme B for n=0:
wherein R1, R2, R3, R4, R5, X, Z, m, p, q and s have the meanings defined above, Y represents an outgoing group such as a chlorine atom, a bromine atom, an RSO3— group wherein R represents a C1-C6 alkyl or a C1-C6 haloalkyl or a phenyl optionally substituted.
The compounds having general formula (I), according to reaction scheme A, for X=nitrogen, can be obtained by condensation of the suitable N′,N′-dialkylamino-N-alkylamine or, for X=sulfur, by condensation of the suitable ω-alkylthioalkylamine with carboxylic acid of a suitable R1 residue, and a condensing agent, optionally in the presence of a base in an organic or aqueous solvent, according to methods well known in the art, for example in Comprehensive Organic Transformations 1989, R. C. Larock, so as to form the corresponding amide.
The intermediate thus obtained is subsequently subjected to alkylation by reaction with the salt of an alkaline metal, such as for example sodium or potassium, of a suitable organic acid having an outgoing group Y, in water or in an organic solvent, at temperatures ranging from room temperature to 100° C., maintaining the pH at values of around 7.5, by the controlled addition of a solution of a strong base.
The compounds having general formula (I), according to reaction scheme B, for X=nitrogen, can be obtained by alkylation of the suitable N′,N′-dialkylamino-N-alkylamine or, for X=sulfur, by alkylation of the suitable ω-alkylthioalkylamine with the desired R1 residue having the outgoing group Y, in the presence of a base in an organic or aqueous solvent, according to methods well known in the art, for example in Comprehensive Organic Transformations 1989, R. C. Larock, so as to form the corresponding tertiary amine.
The intermediate thus obtained is subsequently subjected again to alkylation by reaction with the salt of an alkaline metal, such as for example sodium or potassium, of a suitable organic acid having an outgoing group Y, in water or in an organic solvent, at temperatures ranging from room temperature to 100° C., maintaining the pH at values of around 7.5, by the controlled addition of a solution of a strong base.
The compounds having formula (I), when R1 has the meanings of a C1-C26 alkoxyl group, or a C1-C26 alkylthio group, or a C3-C30 cyclo-alkoxyl group, or a C1-C26 alkylamine group, or a C2-C26 dialkylamine group, can be easily obtained according to reaction scheme C for n different from 0:
wherein R1, R2, R3, R4, R5, X, Z, m, p, q and s have the meanings defined above, Y represents an outgoing group such as a chlorine atom, a bromine atom, an RSO3− group wherein R represents a C1-C6 alkyl or a C1-C6 haloalkyl or a phenyl optionally substituted.
The compounds having general formula (I), according to reaction scheme C, for X=nitrogen, can be obtained by reaction of the suitable N′,N′-dialkylamino-N-alkylamine or, for X=sulfur, by reaction of the suitable ω-alkylthioalkylamine with the desired R1 residue having an alcoholic, or thio-alcoholic, or aminic function when R1 has the meanings of a C1-C26 alkoxyl group, or a C3-C30 cyclo-alkoxyl group, or a C1-C26 alkylthio group, or a C1-C26 alkylamine group, or a C2-C26 dialkylamine group respectively, in the presence of phosgene or one of its functional substitutes, such as, for example, diphosgene, triphosgene, 1,1′-carbonyldiimidazole, in an organic or aqueous solvent, according to methods well known in the art, for example in Comprehensive Organic Transformations 1989, R. C. Larock, so as to form the corresponding carbamate, thiocarbamate or urea.
The intermediate thus obtained is subsequently subjected again to alkylation by reaction with the salt of an alkaline metal, such as for example sodium or potassium, of a suitable organic acid having an outgoing group Y, in water or in an organic solvent, at temperatures ranging from room temperature to 100° C., maintaining the pH at values of around 7.5, by the controlled addition of a solution of a strong base.
The reactions can be conveniently carried out in an aqueous or inert organic solvent, at a temperature ranging from room temperature to the boiling point of the reaction mixture, optionally in the presence of an inorganic or organic base.
Examples of preferred solvents for effecting the reaction are ethers (ethyl ether, isopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, etc.); esters (ethyl acetate, etc.); chlorinated hydrocarbons (methylene chloride, dichloroethane, chloroform, carbon tetrachloride, etc.); aromatic hydrocarbons (benzene, toluene, xylene, etc.); aliphatic hydrocarbons (hexane, heptane, cyclohexane, etc.); aprotic dipolar solvents (N,N-dimethylformamide, dimethylsulfoxide, sulfolane, etc.).
Examples of preferred inorganic bases are: hydroxides, carbonates of alkaline or alkaline earth metals (sodium, potassium, calcium, etc.).
Examples of preferred organic bases are: pyridine, dimethylaminopyridine, aliphatic amines (triethylamine, etc. cyclic amines (morpholine, piperidine, etc.).
If the substituents R1, R2, R3, R4, R5 contain optic or geometric isomerism centres, the compounds having general formula (I) can be present in all possible configurational isomeric forms.
The scope of the present invention therefore also comprises the use of the compounds having general formula (I) as isomeric mixtures in any proportion, and also the formation and use of the single isomers for the control of phytopathogen fungi in the agronomical field.
When deriving from natural extracts, the compounds having general formula (I) can also be present in mixtures of their homologous products and the scope of the present invention consequently also includes the use of the compounds having general formula (I) as mixtures of their homologous products in any proportion, for the control of phytopathogen fungi and bacteria in the agronomical field.
The compounds having general formula (I) can also be present in a hydrated form by the coordination of any number of water molecules, or obtained in aqueous solution and used directly for agronomical purposes.
The compounds having general formula (I) can also contain and possibly coordinate within their structure other metallic cations, such as for example sodium, calcium, potassium, whose number can vary in relation to the preparation method used for the synthesis of the compound having general formula (I).
The scope of the present invention therefore also comprises the use of said solutions of compounds having formula (I), containing said salts for the control of phytopathogen fungi and bacteria in the agronomical field.
The compounds having general formula (I) are capable of controlling numerous fungal and bacterial phytopathogens, also with a reduced sensitivity towards other fungicides.
Examples of phytopathogen fungi and bacteria which can be effectively fought with the compounds having general formula (I) are:
The compounds having general formula (I) are capable of exerting a fungicidal action of both a curative and preventive nature and have a low or zero phytotoxicity.
A further object of the present invention therefore relates to a method for controlling phytopathogen fungi and bacteria in agricultural crops by the application of the amphoteric compounds with a zwitterionic structure of the betainic type having general formula (I) having a direct fungicidal and bacterial activity and a method for the stimulation of the natural defense systems of plants from abiotic stress (temperature, salinity, drought, etc.) and biotic stress and the induction of resistance in the plant itself by the application of the amphoteric compounds with a zwitterionic structure of the betainic type having general formula (I).
The quantity of compound to be applied for obtaining the desired effect can vary in relation to various factors such as, for example, the compound used, the crop to be preserved, the type of pathogen, the degree of infection, the climatic conditions, the application method and the formulation adopted.
Doses of compound ranging from 10 g to 5 kg per hectare generally provide a sufficient control.
For practical uses in agriculture, it is often useful to adopt fungicidal compositions containing one or more amphoteric compounds having a zwitterionic structure of the betainic type having general formula (I).
The application of these compositions can be effected on all parts of the plant, for example on the leaves, stems, branches and roots, or on the seeds themselves before sowing, or on the ground in which the plant grows.
Compositions can be used in the form of dry powders, wettable powders, emulsifying concentrates, microemulsions, pastes, granulates, solutions, suspensions, etc.: the choice of the type of composition will depend on the specific use.
The compositions are prepared in the known way, for example by diluting or dissolving the active substance with a solvent medium and/or a solid diluent, possibly in the presence of surface-active agents.
Solid diluents or supports which can be used are, for example: silica, kaolin, bentonite, talc, infusorial earth, dolomite, calcium carbonate, magnesia, gypsum, clays, synthetic silicates, attapulgite, sepiolite.
Liquid diluents which can be used, in addition to water, are, for example, aromatic organic solvents (xylols or alkyl benzene mixtures, chlorobenzene, etc.), paraffins (oil fractions), alcohols (methanol, propanol, butanol, octanol, glycerin, etc.), esters (ethyl acetate, isobutyl acetate, etc.), ketones (cyclohexanone, acetone, acetophenone, isophorone, ethylamylketone, etc.), amides (N,N-dimethylformamide, N-methylpyrrolidone, etc.).
Surface-active agents which can be used are salts of sodium, calcium, triethylamine or triethanolamine, alkylsulfonates, alkylaryl-sulfonates, polyethoxylated alkylphenols, polyethoxylated esters of sorbitol, ligninsulfonates, etc.
The compositions can also contain special additives for particular purposes, for example adhesion agents such as gum arabic, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylates, etc.
It has also be found, in agronomical practice, that the fungicidal action of compounds having general formula (I) is particularly effective when combined with that of numerous other fungicidal active principles thus creating an excellent instrument for anti-resistance strategies, allowing the applicative doses to be further lowered and stimulating the natural defense of plants.
More specifically, a high synergy has been observed by mixing the compounds having general formula (I) with other compounds also known to be capable of stimulating the natural defense of plants such as salicylic acid, acetylsalicylic acid, copper (II) salt of acetylsalicylic acid ASA2Cu, 2,6-dichloroisonicotinic acid (INA), 1′Smethylester of benzo[1,2,3]thiadiazolyl-7-thiocarboxylic acid (BTH), saccharine, thus enhancing and modulating the biological activity in an effective and safe manner.
In particular, an increased biological activity of the following compounds has been observed:
Said fungicidal compounds are commercial compounds or almost ready to be commercialized.
A description thereof can be easily found in technical literature, for example in “The Pesticide Manual”, 2000, XII edition, British Crop Council Ed., in www. Agrowreports. Com.
IR5885, dipeptide with a fungicidal activity refers to one of the compounds among those claimed in patent application EP 1028125.
An object of the present invention therefore relates to the use of said compositions comprising at least one amphoteric compound having general formula (I) with one or more of the following fungicidal compounds:
Preferred compositions according to the present invention are selected from:
The concentration of active principles in the above compositions can vary within a wide range depending on the active compounds, the applications for which they are destined, the environmental conditions and the type of formulation adopted.
The concentration of active principle generally ranges from 1% to 90%, preferably from 5 to 50%.
The following examples are provided for a better understanding of the invention for illustrative and non-limiting purposes of the present invention.
4.67 g of 3-dimethylamino-1-propylamine are added to a solution of 10 g of lauroylchloride in 50 ml of methylene chloride and 4.74 ml of triethylamine. The mixture is kept under stirring at room temperature for a night. The product obtained is extracted, washed with water, anhydrified with Na2SO4 obtaining, after drying, 12 g of the desired compound (yield: 93%).
Elemental analysis [% found (theoretical)]=C, 71.2 (71.6); H, 12.5 (12.6); N, 9.5 (9.8).
??? of 3-dimethylamino-1-propylamine are added to a solution of 10 g of eicosylbromide in water and 10.5 ml of dimethylamine at 40% in an aqueous solution. The mixture is kept under stirring at room temperature for a night. The product obtained is extracted, washed with water, anhydrified with Na2SO4 obtaining, after drying, 8.1 g of the desired compound (yield: 90%).
Elemental analysis [% found (theoretical)]=C, 80.9 (81.1); H, 14.3 (14.7); N, 4.5 (4.3).
3.41 g of 3-dimethylamino-1-propylamine are added to a solution of 15 g of cholesterylchloroformiate in 70 ml of methylene chloride and 3.49 ml of triethylamine. The mixture is kept under stirring at room temperature for a night. The product obtained is extracted, washed with water, anhydrified with Na2SO4 obtaining, after drying, 15.8 g of the desired compound (yield: 92%).
Elemental analysis [% found (theoretical)]=C, 77.0 (76.8); H, 11.9 (11.2); N, 5.1 (5.4).
12 g of laurylamidopropyl-N,N-dimethylamine in 32 ml of water are charged into a reactor and 4.9 g of sodium monochloroacetate are added. The reaction mixture is slowly heated to 98° C. and the pH is maintained at around 7.5 by the continuous addition of a 50% by weight solution of sodium hydroxide. After about 5 hours the starting products are completely used up and the solution obtained is used as such.
Analogously to what is described in the examples, the following compounds were prepared:
Vine leaves (cultivar Dolcetto), grown in vases in a conditioned environment (20±1° C., 70% relative humidity) are treated by spraying both sides of the leaves with compounds 1, 2 and 3, dispersed in a hydroacetone solution at 20% by volume in acetone.
After remaining 24 hours in a conditioned environment, the plants were sprayed on both sides of the leaves with an aqueous suspension of conidia of Plasmopara viticola (20,000 conidia per cm3).
The plants are kept in a humidity saturated environment at 21° C. for the incubation period of the fungus.
At the end of this period (7 days), the fungicidal activity is evaluated according to an evaluation percentage scale from 0 (completely infected plant) to 100 (healthy plant).
Leaves of wheat plants (cultivar Gemini), grown in vases in a conditioned environment (20±1° C., 70% relative humidity) are treated by spraying both sides of the leaves with compounds 1, 2 and 3, dispersed in a hydroacetone solution at 20% by volume in acetone.
After remaining 24 hours in a conditioned environment, the plants were sprayed on both sides of the leaves with an aqueous suspension of conidia of Erysiphe graminis (200,000 conidia per cm3).
The plants are kept in a humidity saturated environment at a temperature ranging from 18 to 24° C. for the incubation period of the fungus.
At the end of this period (12 days), the fungicidal activity is evaluated according to an evaluation percentage scale from 0 (completely infected plant) to 100 (healthy plant).
Leaves of wheat plants (cultivar Gemini), grown in vases in a conditioned environment (20±1° C., 70% relative humidity) are treated by spraying both sides of the leaves with compounds 1, 2 and 3, dispersed in a hydroacetone solution at 20% by volume in acetone.
After remaining 24 hours in a conditioned environment, the plants were sprayed on both sides of the leaves with an aqueous suspension of conidia of Puccinia recondita (200,000 conidia per cm3).
The plants are kept in a humidity saturated environment at a temperature ranging from 18 to 24° C. for the incubation period of the fungus.
At the end of this period (14 days), the fungicidal activity is evaluated according to an evaluation percentage scale from 0 (completely infected plant) to 100 (healthy plant).
Four-week-old seedlings of arabidopsis thaliana were treated with the compounds having general formula (I) or their mixtures with other fungicides and the leaves were collected after 24 hours of treatment.
The total RNA was extracted from 0.05 g of fresh tissue using the “Genelute mammalian total RNA kit (Sigma)” according to the protocol indications. The cDNA were synthesized using “RevertAid™ M-MuLV Reverse Transcriptase” commercialized by Fermentas Life Sciences according to the following protocol: 2 μg of total RNA were mixed with 0.5 μg of oligo(dT) 18.
Deionized water (nuclease free) was then added to bring the reaction volume to 11 μl, the reaction was subsequently incubated at 70° C. for 5 minutes and then cooled in ice.
The following reagents were then added to the mixture:
4 μl of 5× reaction buffer, 10 mM of dNTP mix, 20 units of Ribonuclease inhibitor.
The reaction was incubated at 37° C. for 5 minutes, 200 units of RevertAid™ M-MuLV Reverse Transcriptase were subsequently added to the mixture and the reaction was incubated at 42° C. for 60 minutes.
The reaction was then blocked by inactivation of the enzyme at 70° C. for 10 minutes.
A quantitative PCR analysis was effected on the cDNA using a mixture of primer/competimers of the ribosomal RNA 18S as internal standard in a ratio of 9:1.
The sequences of the primers used for the PCR reaction are listed below:
The PCR reactions were carried out in 25 μl with the following components:
An additional cycle at 72° C. for 10 min. was subsequently effected.
In
| Number | Date | Country | Kind |
|---|---|---|---|
| MI2005A 001957 | Oct 2005 | IT | national |
| MI2005A 002460 | Dec 2005 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/009813 | 10/10/2006 | WO | 00 | 4/28/2008 |
