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WO1999058725A1 - Procede d'obtention de fragments amplifies d'acide nucleique de particules virales et sous-virales contenant un arn - Google Patents

Procede d'obtention de fragments amplifies d'acide nucleique de particules virales et sous-virales contenant un arn Download PDF

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Publication number
WO1999058725A1
WO1999058725A1 PCT/ES1998/000128 ES9800128W WO9958725A1 WO 1999058725 A1 WO1999058725 A1 WO 1999058725A1 ES 9800128 W ES9800128 W ES 9800128W WO 9958725 A1 WO9958725 A1 WO 9958725A1
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Prior art keywords
viral
pcr
reverse transcription
temperature
reaction
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PCT/ES1998/000128
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English (en)
Spanish (es)
Inventor
Fernando Ponz Ascaso
Vicente Torres Pascual
Begoña BLANCO URGOITI
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Instituto Nacional De Investigacion Y Tecnologia Agraria Y Alimentaria (Inia)
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.)
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Priority to ES09602551A priority Critical patent/ES2121698B1/es
Application filed by Instituto Nacional De Investigacion Y Tecnologia Agraria Y Alimentaria (Inia) filed Critical Instituto Nacional De Investigacion Y Tecnologia Agraria Y Alimentaria (Inia)
Priority to PCT/ES1998/000128 priority patent/WO1999058725A1/fr
Priority to AU70446/98A priority patent/AU7044698A/en
Publication of WO1999058725A1 publication Critical patent/WO1999058725A1/fr

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

  • This invention relates to a method for obtaining amplified fragments of virus nucleic acid with RNA genome and sub-viral particles containing RNA.
  • the resulting amplified fragments can be subsequently analyzed to detect, identify or molecularly typify said viruses and subviral particles.
  • PCR polymerase chain reaction
  • 2 such methods comprise obtaining the amplified nucleic acid fragment and subsequent analysis thereof.
  • the use of spectrophotometric and / or electrophoretic techniques for the analysis of said fragment leads to the detection and / or identification of the pathogen, while the structural analysis of the fragment amplified either by sequencing techniques or by polymorphism analysis techniques of Length of restriction fragments (RFLP) or single chain conformation (SSCP), allows the molecular typing of the pathogen and the conduct of molecular epidemiology studies.
  • RFLP polymorphism analysis techniques of Length of restriction fragments
  • SSCP single chain conformation
  • Some methods for obtaining amplified DNA fragments combine the capture of the pathogen with the enzymatic amplification by PCR.
  • PCR amplification is preceded by a reverse transcription step in order to convert RNA to DNA (substrate for PCR).
  • Methods include performing a step of lysis of the captured viral particle and release of the viral nucleic acid before performing the enzyme amplification or, where appropriate, reverse transcription (e.g., US Patent 5,077). .192), while other methods dispense with said stage of lysis and prior release of nucleic acid (Spanish patents ES 9201232 and ES 9302657).
  • obtaining an amplified nucleic acid fragment comprises: (a) the immunocapture of the pathogen in the wells of a microtiter plate,
  • the present invention provides a solution to the previously raised problems consisting of a process for obtaining amplified fragments of nucleic acid from viral or subviral particles containing RNA, which combines (a) the capture of the pathogen with (b) the addition, in a single operation, of all the reagents involved in the reverse transcription reaction and in the enzymatic amplification reaction by PCR, and with (c) the development of appropriate thermal cycles to perform, in the first place, the transcription reaction Inverse, and secondly, without the need to uncover the container containing the sample to be tested and without the need to add any additional reagents, but simply by modifying the temperature, carry out the PCR enzymatic amplification reaction of the nucleic acid fragment.
  • the amplified fragment can be subjected to spectrophotometric, electrophoretic techniques, or to structural analysis to detect, quantify, identify or molecularly typify the viral or subviral particle in question.
  • This method allows the use of a larger volume of the solution containing the initial viral RNA to perform reverse transcription (typically 80 ⁇ l), that is, A larger part of the sample and the yield and sensitivity increase, the risk of accidental contamination is reduced, the number of sample handling operations is reduced, automation is facilitated and the number of samples to be handled can be increased.
  • Figure 1 shows a photograph of an electrophoresis gel showing the presence of an 879 nucleotide fragment of the potato Y virus genome (PVY), corresponding to the amplified fragment according to the procedure provided by this invention and described in the Example 1.
  • Figure 2 shows a photograph of an electrophoresis gel showing the presence of an 879 nucleotide fragment of the PVY genome, corresponding to the amplified fragment according to the procedure described in Example 1, compared to the same fragment obtained following the procedure described in Spanish patent ES 9201232.
  • the method for obtaining amplified nucleic acid fragments of viral or subviral particles containing RNA present in a sample suspected of containing such particles, object of this invention comprises the steps of: a) adding said sample to the tubes of a thermal cycler and incubating the assembly under suitable conditions to capture said viral or subviral particles in the thermocycler tubes; b) remove the supernatant from the tubes and wash the set of viral or sub-viral particles captured in the tubes of the thermal cycler; c) adding to said tubes, which contain the captured particles, all the reagents necessary to carry out the reverse transcription reaction and the enzymatic amplification reaction by PCR; d) insert the tubes into the thermal cycler; and e) program a suitable thermal cycle in the thermal cycler so that the reverse transcription reaction of the RNA takes place in each tube first, and subsequently the enzymatic PCR amplification reaction of the nucleic acid fragment after thermal deactivation of the reverse transcriptase, for which said thermal cycle comprises: heating the thermocycler tubes at
  • A) Particle capture The first stage of the procedure involves the capture of the viral or subviral particle that contains RNA in the tubes of a thermal cycler.
  • viral particle in the sense used in this description, includes any genome virus.
  • RNA that replicates inside protoplasts, eukaryotic or prokaryotic cells, and at the expense of some enzyme of said protoplasts or cells for example, viruses 7 animals, plant viruses, bacterial viruses and fungal viruses.
  • a particular case of such viral or sub-viral particles is viral or sub-viral pathogens, a term in which viruses with an RNA genome are included and sub-viral particles that comprise RNA that can cause any pathology in animals, including man, or in plants and mushrooms.
  • the tube where the viral or sub-viral particles can be captured is a conventional tube, of those used in the thermal cyclers commonly used to carry out the enzymatic amplification of DNA fragments by PCR.
  • viral or subviral particles will be found in a sample that may be environmental, for example, a water or air sample, or a biological sample, the term of which includes eukaryotic, prokaryotic, protoplasts, mycelium, animal tissues and organs and vegetables, in vitro culture media of any of the above entities, plant or animal systems, plant or animal organisms, as well as any excretion, secretion or transformation product of any of the above entities.
  • a sample may be environmental, for example, a water or air sample, or a biological sample, the term of which includes eukaryotic, prokaryotic, protoplasts, mycelium, animal tissues and organs and vegetables, in vitro culture media of any of the above entities, plant or animal systems, plant or animal organisms, as well as any excretion, secretion or transformation product of any of the above entities.
  • the sample capable of containing them which has previously been homogenized, where appropriate, for example, in a suitable buffer, is added to the tube of the thermal cycler, incubating the assembly at the temperature and during adequate time allowing the capture of said particles.
  • the tube-sample set is incubated at temperatures between 0 and 50 ° C, for a period of time 8 normally longer than 15 minutes, and subsequently the assembly is washed several times with a wash solution, for example, with a saline solution such as phosphate buffered saline (PBS), optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
  • PBS phosphate buffered saline
  • the capture of the viral or subviral particles on the thermocycler tube can be performed by immunocapture by the use of molecules retained on said tube that have affinity for a component or structure related to said particle, for example, antibodies against proteins of the cover of the viral particle, or antibodies against proteins of the cover of a host cell containing the viral particle, or antibodies against double-stranded RNA when it is a viral or subviral particle.
  • the immobilization of molecules with affinity for said viral or subviral particle structures is first performed by any type of physical, chemical or chemical-physical interaction well known to those skilled in the art.
  • said molecules with affinity for a component of the viral or subviral particle are immobilized on the tube of the thermal cycler by adsorption, for which said tube is contacted with a solution of such molecules in a buffer at basic pH, the set is incubated at temperatures between 0 and 50 ° C, for a period of time normally greater than 15 minutes, and the set is washed several times with a wash solution, for example, a saline solution such as PBS, optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
  • a wash solution for example, a saline solution such as PBS, optionally containing a detergent, for example T EEN-20®, buffered at a pH close to neutrality.
  • the immobilized molecules on the tube of the thermal cycler are put in contact with the samples to be analyzed, the whole being incubated under the suitable temperature and time conditions so that said molecules immobilized on the tubes with affinity for such viral or sub-viral particles contained in the sample can immunocapture said particles.
  • the assembly is incubated at a temperature between 0 and 50 ° C, for a period of time normally greater than 15 minutes, followed by several washes with a washing solution, for example, as previously described (PBS + detergent ).
  • the second stage comprises the addition to the tubes of the thermal cycler, which contains the captured viral or subviral particles, of all the reagents necessary for the reverse transcription of the RNA contained in said captured particles and the enzymatic PCR amplification of a DNA fragment previously obtained.
  • the reagents necessary for the reverse transcription reaction comprise a reaction buffer, a reverse transcriptase, an initiating oligonucleotide, a ribonuclease inhibitor, deoxynucleoside triphosphate (dNTPs) and Mg 2+
  • the necessary reagents for the enzymatic amplification reaction by PCR they comprise a buffer for the reaction, a thermostable DNA polymerase, the direct and reverse initiating oligonucleotides, the dNTPs and Mg 2+ .
  • the reagents necessary to carry out the reverse transcription and enzymatic PCR amplification reactions generally comprise: a buffer suitable for performing both reactions;
  • the buffer may be any of the buffers commonly used in reverse transcription and enzymatic PCR amplification reactions, provided that said buffer is suitable for both reactions.
  • Reverse transcriptase can be any enzyme that catalyzes the synthesis of a copy DNA from an RNA that acts as a template.
  • said reverse transcriptase will only be active at the temperature at which the reverse transcription reaction is performed, and advantageously, the reverse transcriptase: (i) will be active at the temperature at which the reverse transcription reaction is performed, (ii ) it may be thermally inactivated and (iii) it will be inactive at temperatures at which the enzymatic amplification reaction is performed by PCR.
  • the reverse transcription reaction is carried out at a temperature equal to or less than 50 ° C, while the PCR enzymatic amplification reaction can be performed at both temperatures equal to or less than 50 ° C. as above 50 ° C, preferably at these latter temperatures. Therefore, in a specific embodiment of this invention the reverse transcriptase: (i) will be active at a temperature equal to or less than 50 ° C, (ii) will be thermally inactivated, and (iii) will be inactive at temperatures above 50 ° C .
  • thermostable DNA polymerase can be used to catalyze the synthesis of a DNA chain from a DNA that acts as a template, at an elevated temperature, such as Taq polymerase, Tfl polymerase, or thermostable DNA polymerases that are activated at temperatures higher than 90 ° C.
  • thermostable DNA polymerase is not active at the temperature at which the reverse transcription reaction is performed, and is only active at the temperature of carrying out the enzymatic PCR amplification reaction.
  • the reverse transcription reaction is typically carried out at a temperature equal to or less than 50 ° C, while the PCR enzymatic amplification reaction can be carried out both at temperatures equal to or below 50 ° C and above 50 ° C, preferably at these last temperatures. Therefore, in a specific embodiment of this invention the thermostable DNA polymerase will be inactive at temperatures equal to or less than 50 ° C.
  • An example of such an enzyme is that sold under the brand name AmpliTaq Gold® (Perkin-Elmer Cetus, United States).
  • an oligonucleotide complementary to an RNA fragment of the viral or subviral particle and of sufficient length for hybridization can be used, in a stable and specific manner, with said RNA fragment.
  • the inverse transcription reaction initiator is the same as the inverse (3 ') initiator that is used in the enzymatic PCR amplification reaction.
  • direct (5 1 ) and inverse (3 ') initiators can 12 appropriate oligonucleotides complementary to DNA sequences derived from the nucleic acid of the viral or subviral particle are used, with a length sufficient for hybridization, in a stable and specific manner, with said DNA sequences and allowing the polymerase to initiate synthesis of the complementary chain.
  • these primers will flank a specific, characteristic or identifying DNA fragment of the family, species or type, of the viral or subviral particle, so that the presence of said amplified fragment is indicative of the existence of the viral or subviral particle in question in the analyzed sample.
  • the reverse initiator (3 ') in the enzymatic PCR amplification reaction is the same as, and acts as, the initiator of the reverse transcription reaction.
  • RNA hydrolysis As a ribonuclease inhibitor, a product capable of inhibiting RNA hydrolysis can be used.
  • An example of such an inhibitor is the human placenta ribonuclease inhibitor (HPRI).
  • the dNTPs include deoxyadenosine triphosphate (dATP), deoxycytosin triphosphate (dCTP), deoxyguanidine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP).
  • dATP deoxyadenosine triphosphate
  • dCTP deoxycytosin triphosphate
  • dGTP deoxyguanidine triphosphate
  • dTTP deoxythymidine triphosphate
  • MgCl 2 magnesium chloride
  • the reagents necessary for performing the reverse transcription and enzymatic PCR amplification steps comprise: a buffer suitable both for the reverse transcription reaction and for the reaction of 13 enzymatic amplification by PCR,
  • a reverse transcriptase activates at a temperature equal to or less than 50 ° C, thermally inactivatable, and inactive at temperatures above 50 ° C;
  • a thermostable DNA polymerase active at temperatures above 50 ° C and inactive at a temperature equal to or less than 50 ° C;
  • the common buffer for both reactions comprises 10 mM Tris-HCl, pH 8.3, 50 mM KC1, the MuLV reverse transcriptase, the human placenta ribonuclease inhibitor, an active DNA polymerase at temperatures above 50 ° C and inactive at temperatures between 37 and 50 ° C, MgCl 2 at a concentration between 1.5 and 5 mM, and a pair of oligonucleotide primers for enzymatic amplification by PCR flanking an identifying region of the viral particle or subviral, of which the reverse initiator oligonucleotide also acts as a reverse transcription initiator.
  • the reagents necessary for the reactions of reverse transcription and enzymatic amplification by PCR are commercial products or, in the case of the initiators, they are oligonucleotides with specific sequences that 14 can be synthesized by conventional techniques. These reagents can be added separately to the thermocycler tubes containing the captured particles or they can be pre-mixed with each other (all or some of them) and form a mixture or cocktail of reagents that is added to such tubes.
  • the third stage comprises introducing the tubes of the thermal cycler containing the viral or subviral particles captured together with the reagents necessary for the reactions of reverse transcription and enzymatic amplification by PCR, and applying to said tubes a suitable thermal cycle to perform First, the reverse transcription reaction, and after completion of this, the enzymatic PCR amplification reaction of the selected nucleic acid fragment, without removing the tube from the thermal cycler, without opening the tubes containing the test samples and with no more than modify the temperature
  • the thermal cycler is programmed to carry out a thermal cycle comprising: heating the thermocycler tubes at a temperature and for a period of time suitable for the reverse transcription reaction to take place, - heating the tubes at a temperature and during a suitable period of time to inactivate reverse transcriptase; Y
  • reverse transcription is performed at the operating temperature of the reverse transcriptase, which, in a particular embodiment of this invention will be equal to or less than 50 ° C, usually between 37 and 50 ° C, for a period of time comprised between 20 and 90 15 minutes .
  • the tubes are then heated to a temperature suitable for thermally denaturing the reverse transcriptase and, if using a thermostable DNA polymerase activatable at elevated temperature, activating said polymerase.
  • the tubes are heated to a temperature greater than 90 ° C, for a period of time between 2 and 10 minutes, whereby the reverse transcriptase is thermally inactivated, the DNA polymerase is activated (if any) and the RNA-DNA hybrid formed is denatured so that the PCR amplification reaction can begin.
  • the enzymatic PCR amplification reaction involves performing an adequate number of repeated heating and cooling cycles, each cycle comprising the steps of:
  • the banding temperature is equal to or greater than 60 ° C
  • the banding and elongation stages can be performed simultaneously.
  • the number of cycles to be performed depends on the degree of amplification of the nucleic acid fragment that is intended to be obtained. In general, adequate results are obtained with a number of cycles between 25 and 50. 16
  • said thermal cycle comprises: a) heating the reaction mixture, formed by the viral or subviral particles captured together with the reagents for the reactions of reverse transcription and enzymatic amplification by PCR, at a temperature between 37 and 48 ° C, for 30 minutes, in order to effect the reverse transcription reaction, then b) heat the reaction mixture at a temperature between 92 and 97 ° C, for 5 minutes, to inactivate the transcriptase conversely, denature the RNA-DNA hybrid and, where appropriate, activate the thermostable DNA polymerase; c) repeat a variable number of times, usually between 25 and 50 times, a heating and cooling cycle, comprising the steps of:
  • - denaturation normally at a temperature greater than 90 ° C, generally between 92 and 97 ° C, for a period of time greater than 15 seconds;
  • - banding at the hybridization temperature of the initiators to the template DNA;
  • - elongation at a temperature between approximately 60 and 94 ° C, for a period of time normally greater than 45 seconds; and d) at the end of the last cycle, maintain the elongation temperature for a period of time normally exceeding 2 minutes in order to terminate the PCR amplification reaction.
  • the fragments thus amplified can be subjected to analysis by spectrophotometric and / or techniques. 17 electrophoretic in order to detect, quantify and / or identify the viral or subviral particle in question, or alternatively they can be subjected to a structural analysis either by nucleic acid sequencing techniques (Maxam, AM, and Gilbert,., (1977 ), Proc. Nati. Acad. Sci. USA, 74: 560; and Sanger, F., et al., (1977), Proc. Nati. Acad. Sci.
  • PVY potato virus Y
  • Samples of potato leaves from healthy plants and plants infected with PVY are homogenized, separately, in a 1/10 ratio (weight / volume) with an extraction buffer composed of 0.5 M Tris-HCl, pH 8, 0, containing 2% polyvinyl pyrrolidone, 1% polyethylene glycol 6000, 0.8% NaCl, 0.005% T EEN-20® (SIGMA CHEMICAL CO., USA) and 0.02% sodium azide.
  • - dNTPs (dATP, dCTP, dGTP and dTTP), [2 ⁇ l of mixture of dNTPs at 10 mM of each one] (equivalent to 0.25 mM), - direct initiator (5 ') 2 ⁇ l to 10 ⁇ M ( 0.25 mM),
  • the initiators used are listed in the section on the LIST OF SEQUENCES, where the direct initiator (5 ') corresponds to the one identified as SEC. ID. N °: 1 and the inverse (3 ') to that identified as SEC. ID. N °: 2.
  • nt corresponding to the capsid protein gene plus the principle of the 3 'non-translatable region (3'-utr).
  • the direct initiator extends from nt 3869 to nt 3891 of the PVY genome, while the reverse initiator extends from nt 4745 to nt 4723.
  • the PVY genome sequence has been described by Hidaka et al., [ Nucleic Acid Research, 20, 3515, (1992)]. For the realization of this Example, 5 different concentrations of MgCl 2 (2, 2.25, 2.5, 2.75 and 3 mM) were tested for comparative purposes.
  • the tubes are then introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 42 ° C, for 30 minutes, with the 19 to carry out the reverse transcription reaction, b) heating at 95 ° C, for 5 minutes, to inactivate the reverse transcriptase and the ribonuclease inhibitor, activate the DNA polymerase, and denature the RNA-DNA hybrid, c) 35 cycles PCR comprising the steps of:
  • the amplified samples were subjected to 0.8% agarose gel electrophoresis in TAE buffer as indicated by Sambrook et al., (1989) [Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor ], were stained with a solution of ethidium bromide (4 ⁇ g / ⁇ l) and photographed with a Polaroid camera on an ultraviolet light transilluminator with an orange filter in front of the lens.
  • MuLV reverse transcriptase 50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) [1 ⁇ l of MuLV reverse transcriptase at 50 units / ⁇ l], - 20 units of HPRI (AMERSHAM, USA), [1 ⁇ l of HPRI at 200 units / ⁇ l ],
  • the tubes are introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 42 ° C, for 30 minutes, in order to carry out the reverse transcription reaction, b) heating at 97 ° C, for 5 minutes, to inactivate the reverse transcriptase and the ribonuclease inhibitor, and denature the RNA-DNA hybrid, 21 c) 35 cycles of PCR comprising the steps of:
  • the reverse transcription reaction is carried out in a final volume of 20 ⁇ l containing:
  • a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KC1, - 5 mM MgCl 2 ,
  • MuLV reverse transcriptase 50 units of MuLV reverse transcriptase (PERKIN-ELMER, USA) and
  • the reverse initiator (3 ') used is the same as in Example 1.
  • the reverse transcription reaction was carried out by heating at 42 ° C for 30 minutes and subsequently 22 tubes were placed on ice.
  • the direct initiator (5 ') used is the same as in Example 1.
  • the tubes are introduced into the thermal cycler where they undergo the following thermal cycle: a) heating at 97 ° C, for 5 minutes, b) 35 PCR cycles comprising the steps of: - denaturation: 20 seconds at 94 ° C;
  • Example 2.1 The products resulting from the amplification reaction from both Example 2.1 and Example 2.2 were subjected to agarose gel electrophoresis as described in Example 1. The results obtained are shown in Figure 2, where it is observed that the yield obtained with the one-step reverse transcription-PCR reaction at a 3 mM concentration of MgCl 2 (Example 2.1: method provided by the invention) is several times higher than that obtained according to the procedure described in the patent 2. 3
  • Example 2.2 Spanish ES 9201232 (Example 2.2).
  • the greater brightness of the visible band in the lane corresponding to the amplified product according to Example 2.1 with a MgCl 2 concentration of 3 mM is indicative of the greater quantity of amplified product.

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Abstract

L'invention concerne un procédé consistant à capturer sur un tube d'un thermocycleur les particules virales ou sous-virales, à ajouter au tube les réactifs nécessaires pour effectuer la transcription inverse (TR) et l'amplification enzymatique par réaction en chaîne de la polymérase (PCR), à introduire le tube dans le thermocycleur et à appliquer un cycle thermique adéquat pour effectuer d'abord la TR puis la PCR sans avoir à ajouter des réactifs entre les deux réactions ou à manipuler les tubes avec échantillons. Pour ce faire, on utilise dans la PCR une polymérase à ADN thermostable. Les fragments amplifiés peuvent être soumis à diverses analyses en vue d'une détection, identification, typification de la particule virale ou sous-virale. Ce procédé trouve une application dans le diagnostic et les études en épidémiologie moléculaire.
PCT/ES1998/000128 1996-12-02 1998-05-08 Procede d'obtention de fragments amplifies d'acide nucleique de particules virales et sous-virales contenant un arn WO1999058725A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
ES09602551A ES2121698B1 (es) 1996-12-02 1996-12-02 Procedimiento para la obtencionn de fragmentos amplificados de acido nucleico de particulas virales y subvirales que contienen rna.
PCT/ES1998/000128 WO1999058725A1 (fr) 1996-12-02 1998-05-08 Procede d'obtention de fragments amplifies d'acide nucleique de particules virales et sous-virales contenant un arn
AU70446/98A AU7044698A (en) 1998-05-08 1998-05-08 Process for obtaining amplified fragments of nucleic acid of viral and sub-viralparticles which contain rna

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES09602551A ES2121698B1 (es) 1996-12-02 1996-12-02 Procedimiento para la obtencionn de fragmentos amplificados de acido nucleico de particulas virales y subvirales que contienen rna.
PCT/ES1998/000128 WO1999058725A1 (fr) 1996-12-02 1998-05-08 Procede d'obtention de fragments amplifies d'acide nucleique de particules virales et sous-virales contenant un arn

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WO1999058725A1 true WO1999058725A1 (fr) 1999-11-18

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0402997A2 (fr) * 1989-06-15 1990-12-19 Akzo Nobel N.V. Procédé pour déterminer des acides nucléiques
WO1991009944A2 (fr) * 1989-12-22 1991-07-11 F.Hoffmann-La Roche Ag Transcriptases inverses obtenues a haute temperature
US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities
ES2044784A1 (es) * 1992-06-12 1994-01-01 Inia Procedimiento para la deteccion e identificacion de patogenos virales y subvirales.
EP0632134A2 (fr) * 1993-07-01 1995-01-04 F. Hoffmann-La Roche Ag Réactif et procédé pour la transcription inverse à haute température suivi par la réaction en chaîne de la polymérase

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU7224591A (en) * 1990-01-24 1991-08-21 United States of America, as represented by the Secretary, U.S. Department of Commerce, The A rapid and sensitive test for detecting hepatitis a virus
JPH04141098A (ja) * 1990-09-29 1992-05-14 Shimadzu Corp Ｒｎａ検出用試薬及びそれを用いたｒｎａの検出方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities
EP0402997A2 (fr) * 1989-06-15 1990-12-19 Akzo Nobel N.V. Procédé pour déterminer des acides nucléiques
WO1991009944A2 (fr) * 1989-12-22 1991-07-11 F.Hoffmann-La Roche Ag Transcriptases inverses obtenues a haute temperature
ES2044784A1 (es) * 1992-06-12 1994-01-01 Inia Procedimiento para la deteccion e identificacion de patogenos virales y subvirales.
EP0632134A2 (fr) * 1993-07-01 1995-01-04 F. Hoffmann-La Roche Ag Réactif et procédé pour la transcription inverse à haute température suivi par la réaction en chaîne de la polymérase

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