WO2009123494A1 - Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique - Google Patents

Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique Download PDF

Info

Publication number
WO2009123494A1
WO2009123494A1 PCT/RU2008/000198 RU2008000198W WO2009123494A1 WO 2009123494 A1 WO2009123494 A1 WO 2009123494A1 RU 2008000198 W RU2008000198 W RU 2008000198W WO 2009123494 A1 WO2009123494 A1 WO 2009123494A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
potato
dna
virus
viral
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/RU2008/000198
Other languages
English (en)
Russian (ru)
Inventor
Юрий Федорович ДРЫГИН
Иосиф Григорьевич АТАБЕКОВ
Ольга Александровна КОНДАКОВА
Сергей Николаевич ЧИРКОВ
Роман Алексеевич ЗИНОВКИН
Валентина Ивановна КИСЕЛЕВА
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to PCT/RU2008/000198 priority Critical patent/WO2009123494A1/fr
Publication of WO2009123494A1 publication Critical patent/WO2009123494A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • C12Q1/6804—Nucleic acid analysis using immunogens

Definitions

  • the invention relates to molecular biology, biotechnology, genetic engineering and medicine and can be used as a universal method for the simultaneous detection of various RNAs in analyzed samples, including for diagnostic purposes in mass analyzes of viral and viroid infections of organisms.
  • RNA sequences in a sample An urgent problem of modern molecular diagnostics is the simultaneous detection of many RNA sequences in a sample.
  • differential analysis of the expression of various transcripts for example, in cells of the same type in normal and pathological conditions, under the influence of various environmental factors, as well as during the natural cycle of the body’s development.
  • Another important aspect is the differential analysis of transcripts in cells of various types.
  • the most efficient technology capable of analyzing multiple nucleotide sequences simultaneously is cellular technology or DNA-chip (microchip) technology (hepe arrau techology, arrau techology, DNA chemistry techology). This technology was developed for genotyping, identification of genomes and organisms, determination of point mutations, molecular diagnosis of infections, oncological diseases by comparing gene expression, etc. It is most widely used in medicine.
  • DNA-chip technology differs in the nature (nucleic, protein, etc.) of the chips and the target and in the methods of signal detection, starting from ordinary fluorescence and ending with the transfer of electronic excitation energy from fluorophores (FRET).
  • FRET fluorophores
  • the advantages of DNA-chip technology consist in automatic application sets of probe oligonucleotides, automatic analysis and determination of the signal-to-noise ratio by fluorescence.
  • DNA microarray technologies are currently used by several large companies - developers and manufacturers of diagnostic kits and DNA chips (Agilept, Herrepdorf, TeleChem Iptepatiopal, Suregrau BIOSCIPSE) and for other commercial companies using other diagnostic companies that use kits .
  • a DNA chip is a flat or bulk substrate, on the surface of which multiple DNA probes from 8 to hundreds of nucleotides in length obtained by chemical synthesis or biochemical (PCR or genetic engineering products) are firmly fixed as dots (like in honeycombs). These DNA are DNA probes that determine the selectivity of binding of those DNA or RNA molecules that are in the analyzed drug. The specificity of binding, in turn, is determined by the complementarity of the heterocyclic bases of the analyzed nucleic acids and the DNA chip, which is detected by hybridization of nucleic acids in the analyzed sample with a DNA probe.
  • nucleic acids in the analyzed preparation are labeled with a fluorescent label, which helps to determine which probe (s) or other (s) nucleic acid (s) are bound.
  • DNA-chip technology in diagnostic analysis is the simultaneous determination of several target pathogens, the primary genome structure of which is known.
  • Another advantage of DNA chips is the ability to detect even single (single nucleotide) differences in polynucleotides.
  • the diagnostic specificity of the combination of oligonucleotides that span the sequence of the detected linear polynucleotide is higher than the hybridization specificity of this polynucleotide.
  • the present application relates to the use of DNA-chip technology and competing technologies for the simultaneous detection of many specific RNA sequences in a sample, in particular for the diagnosis of various viral and viroid infections of farm animals and plant cultures, mainly for the diagnosis of various viral and viral infections of potatoes.
  • the authors first selected the necessary primers for cloning the cDNA of the above viruses in vitro using RT-PCR technology, then the amplified DNA, one strand of which is the cDNA of the target virus, was recombined with plasmid vectors by genetic engineering, recombinant DNAs were expanded ipvivo in E. coli . To create a DNA chip, these plasmids were applied using a robot to cells (cells) on the surface of glass coated with polylysine. To detect viruses in an infected plant, the authors extracted total RNA from the leaf tissue of experimental and control plants.
  • the DNA chip in the cells of which synthetic oligodeoxynucleotides (40-mer) were fixed, complementary RNA of the potato viruses PVA, PVS, PVM, PVX, PVY, PLRV, as well as those capable of distinguishing between the strains of the potato virus Y - PVY N TM and PVY °, was developed by D. Wustriska, O. Lepz, I. Mraz, L. Richerova, S. Kmossh, M. Si ⁇ "Oligopletote-based tisro-arrau: And before it, it is possible to test them. ⁇ eth. 128 (2005) 176-182.
  • RNA preparations were hybridized with an oligo DNA chip.
  • the authors successfully diagnosed both potato infections with one virus and two strains of p ⁇ NTN and ru ⁇ of one and TOGQ of vi py py C a.
  • the specificity of target detection is determined immediately by several oligodeoxynuleotide probes.
  • the sensitivity of determining the target nucleic acid in the standard (described above) DNA chip technology is low (hundreds to thousands of copies), but can be increased to tens of copies using special equipment (powerful photomultiplier) or biochemical signal amplification (using ELISA). Higher sensitivity (Reimap M, Hie C. Dewelortept aux evaletiopof a tiltirlekh RT-PCR fordetiptip taip virises apd and viroid offroato. Atta Vigol.
  • the inventors of the present invention have found that the diene-platinum used previously for DNA labeling is capable, under mild conditions, of binding strongly and quantitatively to RNA.
  • Such diene-platinum-labeled RNA is capable of specifically hybridizing with DNA probes immobilized on a chip. Since the authors found that antibodies to diene-platinized DNA recognize any diene-platinized RNA with approximately the same sensitivity, the resulting heteroduplexes of labeled RNA and corresponding DNA probes can be detected using antibodies to dien-platinized DNA.
  • the present invention relates to a method for simultaneously detecting multiple RNA sequences in a biological sample comprising the following steps: a) providing a DNA chip, which is a substrate with DNA probes immobilized on it, specific for said RNA sequences; b) isolation of total RNA from the sample; c) the introduction of a diene-platinum label into RNA molecules; d) hybridization of the RNA labeled with a diene-platinum tag with a DNA chip under conditions providing for the selective formation of specific heteroduplexes between these RNA sequences and the corresponding DNA probes; e) detection of specific heteroduplexes formed using antibodies to DNA-diene-platinum, and the formation of a specific heteroduplex indicates the presence of the corresponding RNA sequence in the sample.
  • the method provides that, as DNA probes for immobilization on a biochip, recombinant plasmids containing inserts of cDNA fragments of said RNA sequences are used. In yet another embodiment, the method provides that synthetic oligodeoxyribonucleotides specific for said RNA sequences are used as DNA probes for immobilization on a biochip.
  • the present invention relates to a method for simultaneously detecting a plurality of causative agents of viral and / or viroid infections in a biological sample, comprising the following steps: a) providing a DNA chip, which is a substrate with DNA probes immobilized on it, specific for RNA of said causative agents of viral and / or viroid infections; b) the selection of total RNA from the specified sample; c) the introduction of a diene-platinum label into RNA molecules; d) hybridization of the RNA labeled with a diene-platinum tag with a DNA chip under conditions providing the selective formation of specific heteroduplexes of viral and / or viroid RNA and the corresponding DNA probes; e) detection of specific heteroduplexes formed using antibodies to dien-platinum, and the formation of a specific heteroduplex indicates the presence of the corresponding causative agent of viral and / or viroid infection in the sample.
  • the method provides that, as DNA probes for immobilization on a biochip, recombinant plasmids are used containing inserts of cDNA fragments of the diagnosed pathogens of viral and / or viroid infections.
  • the method provides that synthetic oligodeoxyribonucleotides specific for the diagnosed causative agents of viral and / or viral infections are used as DNA probes for immobilization on a biochip
  • the biological sample is a potato tissue sample
  • pathogens are alfalfa mosaic virus, potato virus Y, potato virus M, potato virus S, potato virus A, potato virus Y, potato leaf curling virus, potato tuber spindle viroid, potato virus X.
  • DNA probes immobilized on a DNA chip are recombinant plasmids containing insertion of cDNA fragments of RNAs of said pathogens of viral and viral potato infections, obtained by reverse transcription reaction and PCR using a set of primers SEQ W NO: 1-16 .
  • the present invention relates to a DNA chip for simultaneously detecting a plurality of pathogens of viral and viroid potato infections, where the pathogens are alfalfa mosaic virus, potato virus Y, potato virus M, potato virus S, potato virus A, potato virus Y, twisting virus potato leaves, viroid spindle-shaped potato tubers, potato virus X.
  • the DNA chip according to the present invention is a substrate with DNA probes immobilized on it, representing recombinant plasmids containing inserts of cDNA fragments of RNA of the causative agents of viral and viroid infections of potato obtained by the reverse transcription reaction and PCR using a set of primers SEQ W NO : 1-16.
  • the present invention relates to a set of primers to obtain cDNA fragments of RNA alfalfa mosaic virus, potato virus Y, potato virus M, potato virus S, potato virus A, potato virus Y, potato leaf virus, potato tuber spindle viroid and potato virus X, the sequences of which are presented in SEQ ID NO: 1-16, for the manufacture of a DNA chip according to the present invention.
  • the method can be represented as follows: on a semipermeable cellulose nitrate membrane (DNA chip prototype), solutions of cloned recombinant DNA containing separately inserted cDNA of the seven previously mentioned viruses and viroid are deposited in cells in the form of dots (see Fig. 1) and fixed ultraviolet light. From the analyzed samples, which, as a rule, were a test tube plant or plant leaf tissue (plants such as potatoes, tomatoes, and tobacco were used with equal success), the total cellular RNA was isolated from uninfected (control) and plants infected with phytopathogens.
  • RNA isolation any method known to those skilled in the art can be used to isolate total RNA, for example, using guanidine thiocyanate mixed with phenol (in the English literature TRGZOL Reagept (USPatte No.5346994)), hot water-saturated phenol, phenol with 0.5-4% sodium dodecyl sulfate, chloroform with sodium chloride in a concentration of IM and above (used by the diagnostic company "Agdia", USA). It is preferable to use RNA developed by the authors for RNA isolation. of the present invention, a simple, cost-effective and environmentally friendly method using ammonium trichloroacetate, disclosed in patent application PCTYRU2007 / 000310) ..
  • the authors compared the sensitivity of the diagnostic analysis with DNA probes of different lengths fixed in the DNA chip (see Fig. 4). It turned out that the sensitivity of the determination of pathogens was higher for longer DNA fixed in the cells of the DNA chip.
  • the present invention allows the rapid analysis of multiple potato infections in one experiment, significantly accelerating it in comparison with the traditional method of molecular diagnosis of viroid and viral infections with DNA probes.
  • FIG. 1 Fragments (darkened) of the genomes of potato phytopathogens: alfalfa mosaic virus (BMJI, AMV), potato F virus (FBK), potato virus A (ABK), potato M virus (MBK) and potato virus S (SBK) used to produce cDNA using RT-PCR.
  • BMJI alfalfa mosaic virus
  • FBK potato F virus
  • ABK potato virus A
  • MBK potato M virus
  • SBK potato virus S
  • FIG. 2 Modification with dien-platinum does not alter the electrophoretic mobility of RNA (electrophoresis of the RNA preparation BTM labeled with dien-platinum in 1% agarose gel). About 1 ⁇ g of viral RNA was applied to the gel well).
  • Sample 1 control, unplatinized BTM RNA.
  • Sample 2 platinum RNA BTM. 1 ⁇ g of RNA in a volume of 10 ⁇ l was denatured for 3 min at 70 ° C, 3 ⁇ l of distilled water, 2 ⁇ l of 0.01 M NaClO 4 and 5 ⁇ l of 0.1 M diene-platinum were added. The mixture was incubated for 2 hours at a temperature of 45 ° C. Sample 3 is the same as in sample 2, only the mixture was incubated at 0 ° C.
  • FIG. 3 Antibodies to diene-platinized viral RNAs recognize diene-platinum modified RNA, but do not recognize unmodified RNA.
  • RNA 1-4 modified with diene-platinum: 1.3 - in the ratio of 1 atom of platinum per 10 nucleotides of RNA (1: 10); 2.4 - in a ratio of 1: 5.
  • FIG. 4 Determination of XBK RNA using a DNA microarray.
  • Column 1 of row A, B is a recombinant plasmid containing an insert of full-length XBK cDNA;
  • column 3 of row A, B - ds is a PCR product with a length of 470 nucleotides;
  • column 4 of row A, B — ds is a 321 nucleotide PCR product;
  • column 5 of row A, B is a single-chain (ss) PCR product of 614 nucleotides in length;
  • FIG. 5 The effect of the amount of recombinant plasmid deposited on the membrane on the sensitivity of virus detection using a DNA microarray.
  • Panel D is a healthy plant.
  • FIG. 6 Reuse of the DNA microarray for the determination of viral RNA by the example of XBK cDNA of various lengths. Above is the first use of the microchip, below is reuse.
  • Column 1 of row A, B is a recombinant plasmid containing an XBK cDNA insert;
  • column 2 of row A, B — ds is a PCR product of 614 nucleotides in length;
  • column 3 of row A, B - ds is a PCR product with a length of 470 nucleotides;
  • column 4 of row A, B — ds is a 321 nucleotide PCR product;
  • FIG. 7 Diagnosis of viral and viroid infections of potatoes using a DNA microchip.
  • XBK is the potato X virus.
  • the genomes of viroids, viruses and living organisms are represented by extended polynucleotides (from 246 to tens of billions of nucleotides).
  • the technology of DNA (micro) chips is the most effective for a comprehensive analysis of the genomes of higher organisms, consisting of billions of nucleotides and hundreds of thousands of genes, be it the analysis of mutations, deletions or insertions of nucleotides, or the spectrum of gene expression, a comparative population analysis or analysis of genetic and infectious diseases.
  • DNA microarrays can contain tens of thousands of DNA probes for the simultaneous analysis of thousands of genes or target nucleotide sequences.
  • the present invention provides a new method for simultaneously detecting many different RNA sequences in a sample.
  • the present invention provides a method for simultaneously detecting a variety of pathogens of various viroid and viral diseases of organisms using a DNA chip.
  • the diagnostic capabilities of the method of the present invention are demonstrated by the example of eight economically dangerous for the Russian Federation viral and one viroid infections of an important agricultural crop - potatoes.
  • DNA probes with a length of 20 nucleotides are sufficient to search for a point mutation, and for reliable diagnosis of an infectious disease, it may be insufficient and 40 nucleotides in length. Moreover, for a reliable diagnosis of one infectious agent, even with the simplest genome, several probes that overlap in the sequence of nucleotides are needed.
  • oligodeoxyribonucleotide probes can be carried out on based on the nucleotide sequences of viruses and viroids, which are publicly available, for example, from the GepVapk database. Access to sequences is most conveniently carried out through the EPTRES search system on the website https://www.ncbi.nlm.nih. g Congressv.
  • oligodeoxyribonucleotide probes for immobilization on a DNA chip such factors as specificity, the presence of secondary structures such as hairpins, the presence of non-specific annealing sites on the target sequence, and annealing of primers on themselves and each other are taken into account.
  • oligonucleotide primers for example, the phosphodiester, phosphotriether, phosphite method, etc., but the most widely used method is the phosphite amidite method.
  • the synthesis of primers is carried out using automatic DNA / RNA synthesizers, for example, manufactured by Arrlied BIOSystems (USA).
  • the necessary monomers-synthons, activated resins, devices are manufactured by Arrlied BIOSystems, Glep Research, Ipitogep, Synthol and
  • DNA probes which are recombinant DNA containing inserts of full-sized cDNA or extended cDNA fragments of viroids and / or viruses that cause diseases of crops or animal breeds, rather than short synthetic ones oligodeoxyribonucleotides.
  • DNA probes which are recombinant DNA containing inserts of full-sized cDNA or extended cDNA fragments of viroids and / or viruses that cause diseases of crops or animal breeds, rather than short synthetic ones oligodeoxyribonucleotides.
  • one probe may be sufficient (see, for example: Kondakova O.A., Drygin Yu.F. Diagnosis of potato viroid disease with probes (diene) Pt-DNA, Biotechnology, N ° 4, p. 83 . -90 (1999)]
  • An optimal size in this case is a DNA probe length of at least 300 nucleotides is known that since this length (B. D. Names and SJ Niggips (Editors) Nusleis Asid Nubridizatiop:. a Rrastisal Arrroash, Ohford-Washington DC, 1985, IRL Pgess) melting point and formation duplexes are independent (little dependent) on the length of polynucleotides.
  • Synthetic membranes, gels, activated glass, gold can be used as a substrate, DNA chip material, when analyzed by plasmon resonance, the surface of atomic force microscope probes.
  • Activated substrates commercially available from various manufacturers can also be used for the production of DNA chips (see, for example, Ramakrishpap R, Dorris D, Lublinsky A, Nguyen A, Domanus M, Prokhorova A, Gieser L, Touma E, Lockner R, Tata M, Zhu X, Patterson M, Shirru R, Sendera TJ, Mazumder A.
  • DNA chips can be made by creating a matrix of polyacrylamide gel cells on the surface of the glass substrate, activating the cells, and covalently immobilizing the modified oligonucleotides in the cells that carry active groups (Mirzabekov A. & Kolchinsky A. Macrechi arrlisatiops ip gepomis studios. (2002) In Gepotis Techniques: Resept apt Fitre. Ed. Galas DJ, SJ McCormas. Caister Asadém Res., pp. 163-196).
  • DNA chips can also be made by chemically-induced or photo-induced copolymerization of an oligonucleotide in an acrylamide gel, as previously described (Vasiliskov, V., Timofeev E., Surzhikov S., Drobushev, A., Shisk, V. & Mirzabekova .Fabricatiop of miessorr GmbH Corporation's trademark for a silica gel.
  • the cellulose cellulose membrane which is not the strongest, but widely used in nucleic acid hybridization, was used.
  • the cellulose nitrate membrane (Schleischeg & Schuell, Rotrap BA-85 0.45 ⁇ m or BA 83 0.22 ⁇ m) was soaked in 10 x SSC for 10 min, dried on filter paper.
  • DNA microarrays can be stored for a long time at + 4 ° C.
  • RNAs from infected and uninfected cells were isolated using the method developed by the authors using a non-toxic water-soluble chaotropic agent of ammonium trichloroacetate (see patent application PCT / RU2007 / 000310).
  • a non-toxic water-soluble chaotropic agent of ammonium trichloroacetate see patent application PCT / RU2007 / 000310.
  • To label RNA isolated using a chaotropic agent 1 ⁇ g of RNA in a volume of 10 ⁇ l was denatured for 3 min at 70 ° C, 3 ⁇ l of distilled water, 2 ⁇ l of 0.01 M NaClO 4 and 5 ⁇ l of 0.1 M diene-platinum were added. The mixture was incubated for 2 hours at a temperature of 45 ° C.
  • RNA is significantly less stiffer than those used for labeling with diene-platinum DNA (6O 0 C, 2 hours) [V.I. Kiseleva, M.F. Turchinsky, T.B. Kolesnik, A.M. Attorney, Bioorg. chemistry, v. 20, p. 14-20 (1994); Drygin Yu.F., Musin SM., Kondakova OA, Savenkov E.I., Solomatin CB, Mozheva KA, Acad. PACXH Atabekov I.G.
  • microarray DNA was incubated in a prehybridization solution (5 x SSC, 2x Denhardt solution, 0.1% sodium dodecyl sulfate) for 2 h at a temperature of 65 ° C. Dien-platinized RNA was denatured for 3 min at 7O 0 C and added to the prehybridization solution. Hybridization was carried out in hermetically sealed Petri dishes for 24 h at a temperature of 65 0 C.
  • a prehybridization solution 5 x SSC, 2x Denhardt solution, 0.1% sodium dodecyl sulfate
  • the membranes were washed with Ix SSC with 0.1% sodium dodecyl sulfate and then 0.2 x SSC with 0.1% sodium dodecyl sulfate. Both washes were carried out on a shaker for 15 min at a temperature of 65 ° C. To remove sodium dodecyl sulfate residues, the membranes were washed three times with 0.1 M Tris-HCl buffer solution, pH 9.0, containing 0.14 M NaCl (TBS) for 5 min at room temperature.
  • TSS 0.14 M NaCl
  • an indirect enzyme-linked immunosorbent assay was used on membranes in which primary antibodies were against di-platinized DNA and conjugates of horseradish peroxidase immunoglobulins were used as secondary ones.
  • the membrane was blocked in TBS containing 1% Tween-20 on a shaker for 1 h at room temperature. Excess detergent was removed by rinsing the TBS membrane three times. To detect tags, the membrane was incubated with rabbit affinity purified antibodies to (diene-Pt) DHK. The reaction was carried out in TBS containing 0.5% bovine serum albumin (fraction V), on a shaker for 2 hours at room temperature or overnight at + 4 ° C. Unbound antibodies were removed by washing the membrane three times with TBS containing 0.1% Tween-20 on a shaker for 10 minutes at room temperature.
  • Rabbit antibodies bound to the target were detected using a conjugate of donkey antibodies to rabbit immunoglobulins labeled with horseradish peroxidase (Ammersam).
  • the reaction was carried out in TBS containing 0.5% bovine serum albumin (fraction V) on a shaker for 50 minutes at room temperature. Unbound conjugate was removed by washing the membrane three times with TBS containing 0.1% Tween-20 on a shaker for 10 minutes at room temperature.
  • the membrane was rinsed twice with TBS and incubated for 1-3 min with a chemiluminescent peroxidase substrate (Stag-Gl Gl Chemilumissept Substrate, ISN Biochemilys, Ips.) In accordance with the manufacturer's instructions. Emission was recorded by exposing the membrane with an x-ray film (Kodak MXB FiIm) for 1 to 20 min, depending on the intensity of chemiluminescence. The film was processed in the developer for x-ray films, washed, dried and scanned on an Ersop Regfestiop 1650 scanner (Fig. 4-7).
  • a chemiluminescent peroxidase substrate Stag-Gl Gl Chemilumissept Substrate, ISN Biochemilys, Ips.
  • Emission was recorded by exposing the membrane with an x-ray film (Kodak MXB FiIm) for 1 to 20 min, depending on the intensity of chemiluminescence.
  • the data shown in FIG. 7 show the complete agreement between the results of virus detection by ELISA and using a DNA microarray.
  • a DNA microchip can simultaneously detect a viroid, which could not be detected by enzyme immunoassay due to the natural features of this pathogen.
  • the authors of the present invention set out to show the suitability of the developed diagnostic microchip (prototype DNA chip) for determining viral and viral infections of organisms.
  • Example 1 Platinization of viral RNA and the study of its ability to specific hybridization with a DNA probe.
  • RNA mixture was platinized as described above and used in hybridization with a DNA microarray.
  • a recombinant plasmid was immobilized in the cells of the DNA microarray. containing an insert of XBK cDNA, as well as single-stranded (ss) and double-stranded (ds) fragments of XBK cDNA of various lengths, obtained by polymerase chain reaction. For this, a reverse transcription reaction (AMV RT, Protega) was performed on a matrix of total XBK RNA with scattered seeding (in the presence of random heptamers).
  • Hybridization of platinized RNA with DNA probes specific for potato virus X and identification of hybridization products was carried out as described above (MGA-ELISA technology).
  • Example 2 The study of the influence of the length of DNA probes on the sensitivity of the diagnostic analysis.
  • Potato virus X cDNA inserts or fragments of different lengths were obtained by cloning with vitro symmetric (for double-stranded DNA) or asymmetric (for single-stranded DNA) polymerase chain reaction.
  • the length of the viral cDNA was set by the position of the primers on the templates. As expected (see row B in FIGS. 4 and 6), a significantly greater sensitivity of target RNA detection is obtained with DNA probes containing full-sized XBK cDNA.
  • results obtained indicate that PCR products can also be fixed in the microchip cells; best results, as follows from FIG. 4 were obtained using the longest fragment with a length of 614 nucleotides, which, apparently, is explained by the greater efficiency of hybridization of labeled RNA with a longer cDNA fixed on a DNA microarray.
  • Example 3 The study of the possibility of reuse of DNA microarray to determine viral RNA on the example of XBK cDNA of various lengths.
  • Example 4 Multiple express diagnostics of economically dangerous for the Russian Federation viral and viroid infections of potatoes using DNA microarray.
  • FIG. 7 shows the results of simultaneous molecular diagnostics of the seven most economically dangerous for the Russian Federation viral and viroid infections of potatoes.
  • Alfalfa mosaic virus is common in nature around the world on a wide range of plant species, including potatoes.
  • ELISA enzyme-linked immunosorbent assay
  • the test samples were infected mixed with viruses M and X and separately with the potato virus M.
  • Comparison of the results of diagnostic analysis of DNA-chip and ELISA technologies was evident in favor of the DNA-chip.
  • DNA-chip technology not only confirmed the results of an enzyme-linked immunosorbent assay, but also revealed a viroid infection of the samples, which is not accessible by ELISA.
  • One of the main objectives of the present invention was to develop a diagnostic DNA microarray that allows for the simultaneous detection of viruses and potato viroid in a single sample.
  • This technology for the diagnosis of viral and viroid diseases of plants is based on molecular hybridization of viral RNA labeled with diene PT with complementary DNA immobilized on a cellulose nitrate membrane, followed by the detection of heteroduplexes using ELISA.
  • the DNA microarray allows the determination of viral RNA with a sensitivity of 5 pg RNA of a virus or viroid or more in the analyzed sample of the total RNA preparation obtained from 10-20 mg of potato leaf tissue.
  • the duration of the analysis is no more than 3 days.
  • the shelf life of RNA preparations labeled with dien-platinum and DNA microarrays is at least 6 months at -20 ° C.
  • a DNA microchip can be used for the simultaneous diagnosis of infections of major viruses and potato viroid in unique (for example, collection) variety samples to determine their phytosanitary status, to monitor the effectiveness of recovery from viruses and viroid, etc. It should be emphasized that the use of a DNA microarray makes it possible to increase the efficiency of diagnostic analysis in proportion to the number of cells containing the corresponding recombinant DNA with an insert of a viroid or virus cDNA. Such work can be of particular importance in connection with the start of work to create a genebank of healthy potato varieties in the Russian Federation.
  • DNA probes and a DNA microchip can not only diagnose known viroids, but also with a high probability to identify new, still unknown ones that have similar nucleotide sequences, sufficient for specific hybridization.
  • the developed conditions can be used to create DNA microarrays for the detection of many viroids in the processes of obtaining, maintaining, and certifying healthy planting material for potatoes, hops, citrus fruits, grapes, stone fruits (peach, plum, apricot), pome seeds (apple, pear) , vegetable and flower ornamental crops.
  • the developed DNA microchip for the detection of pathogens of viral and viroid diseases of potatoes can be considered as a prototype DNA microchip for the diagnosis of viroid and viral diseases of any economically important agricultural plant.
  • DNA microarrays similar to the microchip disclosed above can be used to diagnose animal viral infections.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L’invention concerne les domaines de biologie, biotechnologie, génie génétique et médecine et plus particulièrement un procédé universel de détection de différents ARN dans les échantillons analysés, y compris à des fins diagnostiques lors d’analyses en masse d’infections virales ou viroïdes d’organismes vivants. Le procédé prévoit l’extraction de l’ARN global à partir d’un échantillon, le marquage de l’ARN au moyen d’une étiquette diène-platine, l’hybridation de l’ARN étiqueté avec une micropuce qui se présente comme un substrat sur lequel sont immobilisées des sondes ARN spécifiques vis-à-vis des séquences ARN d’intérêt, puis la détection d’hétéroduplex spécifiques au moyen d’anticorps pour le diène-platine d’ADN. L’invention porte sur une micropuce diagnostique destinée à la détermination simultanée de sept infections virales ou viroïdes des pommes de terre dans un échantillon analysé, qui a un impact négatif considérable sur la culture de la pomme de terre en Russie. Selon l’invention, cette technologie de diagnostic moléculaire d’infections virales multiples permet d’accélérer radicalement l’analyse diagnostique d’infections virales mixtes de différents organismes en cas d’épizooties épiphytoties dont le diagnostic visuel apporte des résultats controversés, de déclaration de quarantaine, à des fins de certification de plantes ou animaux agricoles, etc.
PCT/RU2008/000198 2008-04-01 2008-04-01 Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique Ceased WO2009123494A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/RU2008/000198 WO2009123494A1 (fr) 2008-04-01 2008-04-01 Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/RU2008/000198 WO2009123494A1 (fr) 2008-04-01 2008-04-01 Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique

Publications (1)

Publication Number Publication Date
WO2009123494A1 true WO2009123494A1 (fr) 2009-10-08

Family

ID=41135766

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2008/000198 Ceased WO2009123494A1 (fr) 2008-04-01 2008-04-01 Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique

Country Status (1)

Country Link
WO (1) WO2009123494A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5994079A (en) * 1998-02-06 1999-11-30 Digene Corporation Direct detection of RNA mediated by reverse transcriptase lacking RNAse H function
WO2005003318A2 (fr) * 2003-07-02 2005-01-13 Perkinelmer Las, Inc. Analyse et procede de marquage et de detection de microsequences d'arn et de petites sequences de l'arn d'interference
RU2265058C2 (ru) * 2000-06-08 2005-11-27 Ксиао Бинг ВАНГ Способ выявления в образце нуклеиновой кислоты-мишени (варианты)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5994079A (en) * 1998-02-06 1999-11-30 Digene Corporation Direct detection of RNA mediated by reverse transcriptase lacking RNAse H function
RU2265058C2 (ru) * 2000-06-08 2005-11-27 Ксиао Бинг ВАНГ Способ выявления в образце нуклеиновой кислоты-мишени (варианты)
WO2005003318A2 (fr) * 2003-07-02 2005-01-13 Perkinelmer Las, Inc. Analyse et procede de marquage et de detection de microsequences d'arn et de petites sequences de l'arn d'interference

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOONHAM N. ET AL.: "Detection of potato viruses using microarray technology: towards a generic method for plant viral disease diagnosis", J VIROL METHODS., vol. 108, no. 2, March 2003 (2003-03-01), pages 181 - 187 *
BYSTRICKA D. ET AL.: "Oligonucleotide - based microarray: a new improvement in mikroarray detection of plant viruses", J VIROL METHODS., vol. 128, no. 1 - 2, September 2005 (2005-09-01), pages 176 - 82 *
DATABASE GENBANK. 31 October 2007 (2007-10-31), retrieved from http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccor &id=161137770 Database accession no. EU 257478 *

Similar Documents

Publication Publication Date Title
Tsui et al. Molecular techniques for pathogen identification and fungus detection in the environment
US8673595B2 (en) Sample analysis method and assay kit used therein
CA3022666C (fr) Analyses multiplexees d'echantillons d'essai
KR20020008195A (ko) 폴리뉴클레오티드 서열 변형의 미세배열-기초 분석
CN1313908A (zh) 使用可交联的固定的核酸的检测法
JP4256291B2 (ja) 標的核酸配列の検出方法
WO2015076356A1 (fr) Méthode de détection d'arn à chaîne courte
JP2008502367A (ja) オリゴヌクレオチドの高速生成
JP7030051B2 (ja) ２以上の標的核酸を検出するためのプライマーセット、キット及び方法
Baranwal et al. Plant virus diagnostics: traditional to recent and emerging advances
CN104059907A (zh) 含有树状分子染料的寡核苷酸探针及其制备方法和应用
JP2009000099A (ja) 検体中の核酸の検出方法、それに用いるプローブ設計方法、プローブ設計システム
KR102062418B1 (ko) 표적 핵산의 검출 방법
JP2004507207A (ja) サンプル中の生物学的実体を検出するための方法
WO2009123494A1 (fr) Procédé de détection simultanée de différentes séquences arn dans un échantillon biologique
JPS62265999A (ja) 核酸含有試料中の微生物の検出
JP4381457B2 (ja) 標的核酸配列の検出方法
WO2007095055A2 (fr) Procédés et compositions permettant de détecter un acide nucléique viral dans une cellule
US9890418B1 (en) Method of detecting nucleic acids with enhanced signal using dual-functional capture particles
Arya Evolving paradigms in biotechnology for management of crop diseases
RU2816852C1 (ru) Способ пцр-идентификации фитопатогенного гриба fusarium oxysporum
JP7698836B2 (ja) Ｒｔ－ｌａｍｐ法によるサツマイモ病原ウイルスの診断方法の開発
Majumder et al. Molecular approaches for detection of plant pathogens
KR100443569B1 (ko) 길이다형성 ｄｎａ 염기서열을 이용한 다중 증폭중합효소연쇄반응 조건의 최적화에 의한 개인식별 및친자감별 방법
US8518642B2 (en) Method of analyzing probe nucleic acid, microarray and kit for the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08873747

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08873747

Country of ref document: EP

Kind code of ref document: A1