WO1996025909A2 - Procedimientos de amplificacion de genoma y mezclas de oligonucleotidos iniciadores para la deteccion y la identificacion de agentes infecciosos relacionados - Google Patents
Procedimientos de amplificacion de genoma y mezclas de oligonucleotidos iniciadores para la deteccion y la identificacion de agentes infecciosos relacionados Download PDFInfo
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- WO1996025909A2 WO1996025909A2 PCT/ES1996/000031 ES9600031W WO9625909A2 WO 1996025909 A2 WO1996025909 A2 WO 1996025909A2 ES 9600031 W ES9600031 W ES 9600031W WO 9625909 A2 WO9625909 A2 WO 9625909A2
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- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- 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
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- the invention relates to new mixtures of reaction initiators and to new methods of gene amplification useful for the specific detection and identification of related infectious agents, especially infectious agents capable of producing neurological disease and more especially for detection and specific identification of herpes viruses. and human enteroviruses.
- Herpesviridae infectious agents that establish latency after primoinfection causing periodic recurrences under certain conditions. They are enveloped viruses whose genome consists of a double stranded DNA molecule.
- HSV1 Herpes simplex type 1
- HSV2 Herpes simplex type 2
- VZV Varicella zoster virus
- CMV Cytomegalovirus
- HHV6 Human Herpesvirus 6
- EBV Epstein-Barr Virus
- Aseptic meningitis, encephalitis, meningoencephalitis and polyradiculitis are the most common clinical manifestations of central nervous system infections associated with this group of viruses.
- Picornaviridae included in the family Picornaviridae is part of the group of enteroviruses, a genus known since the fourteenth century BC for the production of polio epidemics. Infection by these small non-enveloped viruses and whose genome is constituted by a single-stranded RNA molecule of positive polarity, has a higher incidence in the first years of life and generates a broad spectrum of clinical syndromes. In developed countries they are the most frequent etiologic agents of cases of aseptic meningitis, a neurological syndrome that in Spain is associated with enteroviruses in 90% of the total cases depending on the year and the epidemic outbreaks produced.
- enteroviruses and herpes viruses are capable of producing neurological syndromes in both immunocompetent and immunocompromised patients.
- Aseptic meningitis due to its high frequency, in addition to encephalitis and meningoencephalitis, due to its worse clinical evolution, are the reason for different studies aimed at finding the viruses involved in its production.
- the control of acute neurological diseases can be done by establishing an effective diagnosis that allows the finding and identification of the etiologic agent associated with a specific neurological picture.
- the detection and identification of these infectious agents is one of the fundamental needs of a microbiological diagnostic laboratory.
- laboratory diagnosis is based on the isolation of different viruses in cell cultures. This technique is slow and It also requires an additional effort to maintain different cell lines in order to cover the largest number of enterovirus serotypes, in addition to the different herpes viruses.
- a positive result in isolation can be established in a period of time between 4 and 15 days.
- certain enteroviruses are unable to grow in cell cultures, which prevents their detection in this type of diagnostic system.
- the indirect systems of analysis of specific antibodies produced against a certain herpes virus are very useful when analyzing samples where the antibody titer is high. This situation is different in those cases of neurological infection, where the sample analyzed is mainly cerebrospinal fluid (CSF). In these types of samples the specific antibody titer against a virus can be very low and barely detectable.
- CSF cerebrospinal fluid
- indirect methods of analysis of specific antibodies against enteroviruses do not have a diagnostic value due to their free circulation as normal pathogens in the healthy population.
- the great antigenic diversity of enteroviruses does not. allows to develop sufficiently sensitive and specific serological techniques to identify all types.
- PCR genomic amplification techniques
- Viruses belonging to the same family and that are intrinsically related to each other have a high degree of homology of certain genomic areas; for example, the 5 'non-coding region, in the case of enteroviruses and the region that codes for the DNA polymerase gene, in the case of Herpes viruses.
- genomic areas for example, the 5 'non-coding region, in the case of enteroviruses and the region that codes for the DNA polymerase gene, in the case of Herpes viruses.
- These highly conserved genomic areas are the subject of molecular studies and in them common and specific primers are designed that will be used in the PCR technique to be able to detect and identify each virus in a single assay.
- the main advantage of this type of trials is the saving of clinical sample and time to establish a differential etiological diagnosis.
- multiple amplification techniques not only allows detecting any enteroviruses or herpesviruses including those that are difficult to grow, but also allows their specific identification in the case of herpes viruses, even if they are present in quantities as small as in CSF. It should be noted that a multiple amplification technique also presents a series of problems and problems that are difficult to solve. The problems of nonspecificity, reproducibility and lack of sensitivity are, among others, issues that a priori, are necessary to consider, especially at the time of the design of the specific initiators of each virus to be amplified. Once the primers have been designed, the standardization phase of the multiple amplification technique is essential, and it is during this period that they present the greatest incompatibility problems at all levels.
- the present invention aims to describe new mixtures of reaction initiators and new Genomic amplification procedures useful for the specific detection and identification of related infectious agents, especially infectious agents capable of producing neurological disease and more especially for specific detection and identification of human herpesviruses and enteroviruses, viruses without any significant homology at genomic level.
- An aspect of the invention is a genome amplification method for the detection of related infectious agents in a single reaction mixture, characterized by
- oligonucleotide initiator mixtures each mixture being specifically designed for a family of infectious agents with genomically related sequences and characterized in turn by: i) obtained as a simple sum of oligonucleotides, ii) to include, each of the component oligonucleotides of said mixture, preferably at its 3 'end, homologous sequences selected from the related sequences to be amplified, iii) by being able to also include each of the component oligonucleotides of said mixture, preferably at its 5' end, non-homologous sequences selected from the related sequences to be amplified, and iv) because one or more of the component oligonucleotides of said mixture can be distinguished from known sequences that are to be amplified in at least one nucleotide, and
- homology in the sense used in this description, refers to nucleotide sequences that have a homology to each other greater than
- Another aspect of the invention are mixtures such as those described for the above procedure.
- the improvement in specificity is achieved by increasing, if necessary, the length of the initiators and extending them at their 5 'end towards unconserved areas between the different family members and thus achieving a sufficient initiator length to avoid nonspecific hybridization. with similar sequences that may be present in genomes with high complexity, such as the human.
- the mixtures and procedures described above are useful for the detection of infectious agents that produce similar pathologies but are not genomically related to each other, especially those that cause pathology in humans, such as those belonging to the Herpesviridae and Picornaviridae families.
- Another aspect of the invention is a method of detecting and identifying related genomic sequences, characterized by
- Another aspect of the invention is a genome amplification method for the detection and identification of related infectious agents in a single reaction mixture, characterized in that at least one of the initiators used is a characterized mixture.
- Another aspect of the invention are mixtures such as those described for the above procedure.
- Another aspect of the invention is a method of detecting and identifying related genomic sequences, characterized by
- This procedure also allows the detection and identification of related infectious agents, in two consecutive reactions.
- the complex mixture of initiators used in the second reaction does not produce nonspecific amplifications due to its use in presence of a complex genome. This is achieved using the reaction product obtained in the first amplification reaction as the substrate of the second reaction.
- the mixtures and procedures described above are especially useful for the detection and specific identification of different infectious agents related to produce similar pathology, especially in man.
- Another aspect of the invention is the inclusion of an internal amplification control in the above procedures and mixtures that are used for the detection and identification of related infectious agents.
- the above processes and mixtures can include in the reaction mixture itself specific amounts of a genome that cannot be expected in a clinical sample, in addition to the reaction initiators or other specific reagents that allow its detection if the reaction amplification has developed in the absence of inhibitors.
- the inclusion of the internal amplification control will allow the detection of samples containing amplification inhibitors, thus easily identifying false negative results.
- a final aspect of the invention are mixtures of oligonucleotides designed to be used as initiators of the amplification reaction, characterized in that at least one of the component oligonucleotides of said mixture differs from the known sequences that are to be amplified in at least one nucleotide.
- the alteration of the sequence according to the invention has as main objective to make the complex mixtures of initiators compatible with each other; that is, no hybridization occurs between the different components of the oligonucleotide mixtures present in the amplification reaction mixture.
- the oligonucleotide hybridizations, frequent in complex mixtures, produce during the amplification reaction sequestration of the necessary reagent and, frequently, the formation of initiator dimers.
- An embodiment of the invention is the application of the mixtures and methods described above for the detection and identification of related infectious agents for being able to produce similar pathology, especially those capable of producing similar pathology in man, more especially those capable of producing disease.
- neurological in man specifically those belonging to the Herpesviridae and Picornaviridae families.
- FIG. 1 Schematic representation of the alignments of the nucleotide sequences corresponding to: (A) target fragments of the DNA polymerase genes of the 6 human herpesviruses: Herpes simplex type 1 (HSV1); Herpes simplex type 2 (HSV2); Varicella-zoster virus (VZV); Cytomegalovirus (HCMV); Human herpesvirus 6 type A (HHV6-A); Epstein-Barr virus (EBV); of human DNA polymerase (HUMAN) and Swine Pseudorrabia Virus (PRV), (B) Oligonucleotides initiating the first amplification reaction and (C) Oligonucleotides initiating the second amplification reaction. Each oligonucleotide is identified with the sequence code (SEQ ID No :).
- Figure 3 Figure 3
- Molecular weight marker M
- Coxsackievirus A 16 (lane 1); HSV1 (lane 2); HSV2 (lane 3); VZV (lane 4); CMV (lane 5); HHV6 (lane 6); EBV (lane 7); Mix of all herpesviruses (lane 8); Human fetal fibroblasts (lanes 9 and 17); Coxsackievirus A16 and HSV1 (lane 10); Coxsackievirus A16 and HSV2 (lane 11); Coxsackievirus A16 and VZV (lane 12); Coxsackievirus A16 and CMV (lane 13); Coxsackievirus A16 and HHV6 (lane 14); Coxsackievirus A16 and EBV (lane 15); Coxsackievirus A16 and mixture of all herpesviruses (lane 16).
- the 500 bp fragment corresponds to the enteroviruses and the 194 bp fragment to the herpesviruse
- FIG. 4 Agarose gel electrophoresis of the amplification fragments generated in Example 4.
- Molecular weight marker (M) molecular weight marker of the amplified fragments: 120 bp HSV1 and HSV2, 98 bp VZV, 78 bp CMV, 66 bp HHV6 and 54 bp EBV (lanes 1, 10 and 19).
- HSV1, HSV2, VZV, CMV, HHV6, EBV (lanes 2, 3, 4, 5, 6 and 7 without Coxsackievirus A16 RNA respectively, lanes 11, 12, 13, 14, 15 and 16 in the presence of Coxsackievirus A16 RNA respectively).
- Mixture of all herpesviruses (lane 8 and 17 with and without Coxsackievirus A16 RNA respectively); Human fibroblasts (lanes 9 and 18).
- Molecular weight marker (M) 500 bp fragment corresponds to the amplification of Coxsackievirus A16 (lanes 1, 3 and 4 ), the 194 bp fragment corresponds to the first PRV amplification (lanes 2, 3 and 4).
- M Molecular weight marker
- HSV1, HSV2, VZV, CMV, HHV6, EBV and human fetal fibroblasts initiators of the second herpesvirus multiple amplification reaction (lanes 1 to 8 respectively), with PRV initiators in addition to those of multiple amplification of herpesvirus (lanes 9 to 16 respectively), with PRV primers and PRV DNA (lanes 17 to 24 respectively).
- Figure 7 Agarose gel electrophoresis of the amplification fragments generated in the first and second amplification according to Example 10.
- Molecular weight marker M
- 500 bp fragment for Coxsackievirus A16 lane 1
- the fragments 194 bp for HSV1 lanes 2 and 3)
- HSV2 lane 4
- a method for differential diagnosis of related infectious agents is provided by their ability to produce a similar pathology, belonging to different families that are not genomically related to each other, which includes (a) the detection of said infectious agents by enzymatic amplification of a fragment of their genomes and (b) the identification of said agents infectious from the products of the amplification reaction, wherein said amplification reaction is carried out in a single reaction mixture comprising, as the reaction initiator, a combination of oligonucleotide mixtures, each of which It has been specifically designed for each of the families of infectious agents to be detected so that each oligonucleotide mixture: i) is obtained as a simple sum of oligonucleotides, ii) each of the component oligonucleotides of said mixture, preferably includes 3 ' end, homologous sequences selected from the genomically related sequences that are to be amplified, üi) each of the component oligonucleotides of said mixture, may
- a specific application of this method consists in the detection and identification of infectious agents that produce a similar pathology in humans, in particular, a neurological pathology, such as that caused by infectious agents belonging to the Herpesviridae families and
- Picornaviridae particularly that caused by human herpesviruses and enteroviruses.
- the mixture for the amplification reaction further includes an internal amplification control comprising the genome of a virus that should not be present in the sample to be studied together with specific reaction initiators of a fragment of said genome.
- said genome is derived from a viral species belonging to one of the virus families to be identified and is not capable of infecting the animal species, for example the human one, on which the diagnosis is made.
- said genome corresponds to a herpesvirus that cannot infect man such as porcine herpesvirus 1.
- the identification of the infectious agent once detected can be done either by hybridization of the amplified sequences with specific oligonucleotide probes or mixtures of specific oligonucleotides of the species or genera to be identified or, alternatively, by means of a second amplification reaction of the resulting products of the first amplification reaction in which the reaction mixture for said second amplification reaction comprises, as a reaction initiator, a mixture of oligonucleotides designed such that: i) is obtained as a simple sum of oligonucleotides, ii) each of the component oligonucleotides of said mixture, preferably includes at its 3 ' end, specific sequences of each of the sequences to be typified in said second amplification reaction, iii) the specific amplified fragments are different from each other by size or by different marking physical or chemical of each d and the component oligonucleotides of said mixture, and iv) one or more of the component oligonucleot
- the proposed method also contemplates the possibility of confirming the Identity of each virus species identified by an additional specific hybridization reaction with a specific oligonucleotide of each species whose identity is to be confirmed or alternatively by an additional genomic amplification reaction using the appropriate initiator oligonucleotides.
- the diagnostic method facilitated by this invention specifically identifies the presence of herpes simplex virus (type 1 or type 2) in a sample (see Example 10).
- Example l Design of primers for the detection of human enteroviruses in order to obtain a multiple method of amplification of enteroviruses and / or herpesviruses.
- the genus enterovirus encompasses a very large number of viruses from which only a minority have been able to obtain complete sequences of their genome.
- the 5 ' non-coding region (5 NCR) has been highlighted by the enormous homology presented by sequenced enteroviruses and whose sequences are available to date in the GENBANK database.
- the 5 ' NCR region has an enormous biological interest for its participation in the beginning of the replication of this group of viruses.
- SEQ ID No: 2 NEGATIVE POLARITY SEQ ID No: 3
- the degree of homology between the two designed primers and the corresponding specific sequence of each of the enteroviruses whose genome has been sequenced is very high, becoming exactly the same in most viruses. This high degree of homology allows very specific and highly sensitive enteroviru ⁇ amplification reactions.
- the system must be made compatible in order to amplify in a first RNA amplification reaction from the enteroviruses, in addition to DNA from the herpesviruses.
- a reaction mixture has been designed containing the first reaction primers of enterovirus and herpesvirus amplification.
- Herpesvirus primers were designed and described in Spanish Patent P9201174.
- the mixture of initiators consists of:
- Example 2 Detection of human enteroviruses and / or herpesviruses in a first amplification reaction.
- the samples to be analyzed contain different dilutions of enteroviruses mixed with a certain amount of human fibroblasts.
- the method of preparation of viral RNA has been described above (Casas, I. Powell L., Klapper PE, Cleator GM A new extraction method for RNA and DNA by precipitation in clinical samples. J. Virol. Methods 1994, in press). Once the nucleic acids (DNA and RNA) present in the sample to be precipitated are diluted in 10 ⁇ l of water.
- RNA present in 5 ⁇ l of the previous dilution is transformed into cDNA by a 30-minute reverse transcription at a temperature of 37 ° C, under the conditions recommended by the manufacturer of the reverse transcriptase, a vital enzyme for this process. 100 pmol of negative polarity initiator is used, since the RNA strand has a positive polarity.
- the cDNA will undergo an amplification of 40 cycles, with an initiator hybridization temperature of 60 ° C.
- the denaturation temperature range is set between 92 ° C and 95 ° C, although 94 ° C has preferably been chosen.
- the extension temperature of the DNA polymerase is set between 60 ° C and 80 ° C, although preferably it is 72 ° C.
- the reaction mixture is constituted by a mixture of the two initiator mixtures in a chosen concentration of 100 pmol of each of them.
- the total volume chosen from the reaction is 50 ⁇ l.
- Example 3 Design of primers for the specific detection of enteroviruses and / or herpesviruses in which the marking is the molecular weight of the amplified fragment
- the primers For the identification of the fragments generated in Example 2, the primers have been designed for a second amplification reaction that uses the fragment obtained in the first amplification reaction as the target molecule. This increases the sensitivity and specificity of the identification reaction. From the related and compared sequences in Figure 1, they have been initially selected the following common primers for all enteroviruses
- Example 4 Detection and identification by molecular weight of the amplified fragment of human herpesviruses in the presence of enteroviruses.
- Example 5 Common and specific probe design for the identification of human enteroviruses.
- Example 6 Design of the primers for the detection of an internal amplification control in each reaction tube.
- the multiple method of amplification of herpesviruses already described in Spanish Patent P9201174 has been taken as the basis of the design. It is based on the multiple amplification of the herpesviruses HSV1, HSV2, VZV, CMV, HHV6 and EBV using a mixture of primers characterized in said patent because their 3 ' ends are conserved areas between the DNA polymerase genes of human herpesviruses.
- primers capable of reamplifying the fragment generated in the first PRV amplification reaction have been designed as an internal amplification control.
- the following primers have been designed for the second amplification reaction:
- the specific PRV fragment would have a size of 140 bp after the second amplification so it is easily detectable.
- Example 7 Detection of herpesvirus in the presence of the specific primers of the PRV virus used as internal control of first and second amplification.
- Example 4 According to the procedure described in Example 4, a new mixture of specific herpes virus initiators has been used which also contains the specific PRV initiators, used as an internal amplification control. First, they have undergone a first DNA amplification reaction of each of the herpes viruses according to the procedures described in Example 1 and using the compound initiator mixture by:
- the DNA fragments from the first amplification reaction corresponding to each of the herpes viruses (HSV1, HSV2, VZV, CMV, HHV6 and EBV) have been amplified ). It has been possible to detect specific DNA from each of the herpes viruses and PRV after the second amplification reaction using the mixture of specific primers consisting of:
- Each virus is identified based on the molecular weight of the amplified fragments Figure 6.
- Example 8 Detection of enteroviruses and herpesviruses in the presence of specific PRV virus primers as internal control of first and second amplification reactions.
- an internal amplification control the one designed in Example 6 has been used.
- the mixture of primers consisting of those specific for enteroviruses and those of herpesviruses is enriched by the presence of the initiators of the internal control, which consists of a cloned fragment of the gene. of the PRV virus polymerase DNA.
- Example 9 Design of the mixture of HSV1 and HSV2 specific initiators The homology between herpes simplex type 1 virus and herpes simplex type 2 virus is very high. Similar to how the design of the primers of Example 6 has been performed, the nucleotide sequences of the HSV1 and HSV2 specific primers have been determined for a second amplification reaction in which the target molecule is the resulting amplification fragment of a first amplification reaction previously developed and described in Spanish Patent P9201174. From the aligned and compared sequences of The human herpesviruses of Figure 2 have designed specific primers for these two viruses so homologous.
- Amplification using the designed mixtures should generate fragments of 93 bp for HSV1 and 138 for HSV2.
- Example 10 Detection of specific sequences of HSV1 or HSV2 in samples containing or not enterovirus RNA.
- TYPE nucleic acid
- C STRANDEDNESS: single
- D TOPOLOGY: linear
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL YES
- ANTI-SENSE NO (vi) ORIGINAL SOURCE:
- ORGANISM Herpes simplex virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGCATCATCT ACGGGGACAC GGA 23
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL YES
- ORIGINAL SOURCE
- ORGANISM Varicella-zoster virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
- MOLECULE TYPE DNA (genomic)
- Üi HYPOTHETICAL: YES
- ORGANISM Human cytomegalovirus
- ORGANISM Human herpesvirus 6
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAGGTAATTT ATGGTGATAC GGA 23
- MOLECULE TYPE DNA (genomic)
- ORGANISM Varicella-zoster virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
- MOLECULE TYPE DNA (genomic)
- Üi HYPOTHETICAL: YES
- ORGANISM Human cytomegalovirus
- ORGANISM Human herpesvirus 6
- SEQUENCE DESCRIPTION SEQ ID NO: 12: TGTCTACCAA TGTATCTTTT TTT 23
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE cDNA to mRNA
- HYPOTHETICAL YES
- ANTI-SENSE NO
- ORIGINAL SOURCE
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL YES
- ORGANISM Varicella-zoster virus
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 17: TGAGGGGATA GCTAAAATCG 20
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- Üi HYPOTHETICAL: YES
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL YES
- ANTI-SENSE NO
- ORGANISM Herpes simplex virus
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 21: GGTGAACGTC TTTTCGAACT C 21 (2) INFORMATION FOR SEQ ID NO: 22:
- ORGANISM Varicella-zoster virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
- ORGANISM Human cytomegalovirus
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 23: GACGAAGACC TTTTCAAACT C 21
- ORGANISM Pseudorabies virus
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 27: CGCGTGGTCT ACGGGGACAC GGA 23
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL YES
- ORGANISM Pseudorabies virus
- xi SEQUENCE DESCRIPTION: SEQ ID NO: 29:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL YES
- ORGANISM Herpes simplex virus type 1
- ORGANISM Herpes simplex virus type 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GACACGGACT CCATTTTCGT TT 22
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU64614/96A AU6461496A (en) | 1995-02-17 | 1996-02-16 | Process for the genome amplification and mixtures of induceroligonucleotides for the detection and identification of related infectious agents |
GB9621809A GB2301888A (en) | 1995-02-17 | 1996-02-16 | Process for the genome amplification and mixtures of inducer oligonucleotides for the detection and identification of related infectious agents |
EP96901812A EP0789081A2 (en) | 1995-02-17 | 1996-02-16 | Process for the genome amplification and mixtures of inducer oligonucleotides for the detection and identification of related infectious agents |
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ESP9500320 | 1995-02-17 | ||
ES09500320A ES2093554B1 (es) | 1995-02-17 | 1995-02-17 | Procedimientos de amplificacion de genoma y mezclas de oligonucleotidos iniciadores para la deteccion y la identificacion de agentes infecciosos relacionados. |
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WO1996025909A2 true WO1996025909A2 (es) | 1996-08-29 |
WO1996025909A3 WO1996025909A3 (es) | 1996-09-26 |
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EP (1) | EP0789081A2 (es) |
AU (1) | AU6461496A (es) |
CA (1) | CA2188134A1 (es) |
ES (1) | ES2093554B1 (es) |
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EP1618214A2 (en) * | 2003-04-25 | 2006-01-25 | Becton, Dickinson and Company | Detection of herpes simplex virus types 1 and 2 by nucleic acid amplification |
EP1659187A1 (en) * | 2004-11-18 | 2006-05-24 | bioMerieux B.V. | Nucleic acid sequences that can be used as primers and probes in the amplification and detection of HSV DNA and method for the amplification and detection of HSV DNA using a transcription based amplification |
WO2009122201A1 (en) * | 2008-04-03 | 2009-10-08 | Genomica S.A.U. | Method for detection of herpesvirus in a test sample |
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Publication number | Priority date | Publication date | Assignee | Title |
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DE19921419A1 (de) | 1999-05-08 | 2000-11-16 | Univ Ruprecht Karls Heidelberg | Verfahren zum spezifischen Nachweis und zur Identifizierung retroviraler Nukleinsäuren / Retroviren in einem Untersuchungsgut |
SE0102198D0 (sv) * | 2001-06-20 | 2001-06-20 | Gun Frisk | New sequences |
WO2003051388A2 (en) * | 2001-12-18 | 2003-06-26 | Mondobiotech Laboratories Anstalt | Pharmaceutical composition of interferon gamma or pirfenidone with molecular diagnostics for the improved treatment of interstitial lung diseases |
EP1430902A1 (en) * | 2002-12-20 | 2004-06-23 | Mondobiotech Laboratories Anstalt | Pharmaceutical composition of interferon gamma with molecular diagnostics for the improved treatment of asthma bronchiale |
DE10320519A1 (de) * | 2003-04-30 | 2004-11-25 | 4Base Lab Gmbh Advanced Molecular Analysis | Verfahren zum Nachweis infektiöser (+)-Strang-RNA-Viren, insbesondere infektiöser Enteroviren |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991002091A1 (en) * | 1989-08-10 | 1991-02-21 | Northwestern University | Method of identifying herpesviruses and oligonucleotides for use therein |
WO1991010675A1 (en) * | 1990-01-19 | 1991-07-25 | Stichting Researchfonds Pathologie | Primers and process for detecting human papillomavirus genotypes by pcr |
US5075212A (en) * | 1989-03-27 | 1991-12-24 | University Of Patents, Inc. | Methods of detecting picornaviruses in biological fluids and tissues |
WO1993025707A2 (es) * | 1992-06-05 | 1993-12-23 | Instituto De Salud Carlos Iii | Procedimientos de amplificacion de genoma y mezclas de oligonucleotidos iniciadores para la deteccion y la identificacion de secuencias genomicas relacionadas |
WO1994004706A1 (en) * | 1992-08-24 | 1994-03-03 | Akzo Nobel N.V. | Elimination of false negatives in nucleic acid detection |
US5354653A (en) * | 1991-02-25 | 1994-10-11 | Iatron Laboratories, Inc. | Method of type-specific detection of herpes simplex virus |
EP0628640A1 (en) * | 1993-06-04 | 1994-12-14 | Becton, Dickinson and Company | Simultaneous amplification of multiple targets |
-
1995
- 1995-02-17 ES ES09500320A patent/ES2093554B1/es not_active Expired - Fee Related
-
1996
- 1996-02-16 WO PCT/ES1996/000031 patent/WO1996025909A2/es not_active Application Discontinuation
- 1996-02-16 CA CA002188134A patent/CA2188134A1/en not_active Abandoned
- 1996-02-16 EP EP96901812A patent/EP0789081A2/en not_active Withdrawn
- 1996-02-16 GB GB9621809A patent/GB2301888A/en not_active Withdrawn
- 1996-02-16 AU AU64614/96A patent/AU6461496A/en not_active Abandoned
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5075212A (en) * | 1989-03-27 | 1991-12-24 | University Of Patents, Inc. | Methods of detecting picornaviruses in biological fluids and tissues |
WO1991002091A1 (en) * | 1989-08-10 | 1991-02-21 | Northwestern University | Method of identifying herpesviruses and oligonucleotides for use therein |
WO1991010675A1 (en) * | 1990-01-19 | 1991-07-25 | Stichting Researchfonds Pathologie | Primers and process for detecting human papillomavirus genotypes by pcr |
US5354653A (en) * | 1991-02-25 | 1994-10-11 | Iatron Laboratories, Inc. | Method of type-specific detection of herpes simplex virus |
WO1993025707A2 (es) * | 1992-06-05 | 1993-12-23 | Instituto De Salud Carlos Iii | Procedimientos de amplificacion de genoma y mezclas de oligonucleotidos iniciadores para la deteccion y la identificacion de secuencias genomicas relacionadas |
WO1994004706A1 (en) * | 1992-08-24 | 1994-03-03 | Akzo Nobel N.V. | Elimination of false negatives in nucleic acid detection |
EP0628640A1 (en) * | 1993-06-04 | 1994-12-14 | Becton, Dickinson and Company | Simultaneous amplification of multiple targets |
Non-Patent Citations (5)
Title |
---|
ACTA OTOLARYNGOL., vol. 514, 1994, páginas 132-134, XP002009399 TOSHIYA AONO ET AL.: "Detection of human alpha-herpesvirus DNA using consensus primers and specific probes" * |
JOURNAL OF CLINICAL MICROBIOLOGY, vol. 32, num. 2, 1994, páginas 285-291, XP002009400 KAMMERER ET AL.: "Nsted PCR for specific detection and rapid identification of human picornaviruses" * |
JOURNAL OF VIROLOGICAL METHODS, vol. 30, 1990, páginas 215-227, XP000319809 VANDENVELDE ET AL.: "Fast multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis" * |
JOURNAL OF VIROLOGICAL METHODS, vol. 44, 1993, páginas 261-269, XP002009397 TENORIO ET AL.: "Detection and typing of human herpesvirus by multiplex polymerase chain reaction" * |
MED. MICROBIOL. IMMUNOL., vol. 179, 1990, páginas 177-184, XP002009398 KIMURA ET AL.: "Detection and direct typing of herpes simplex virus by polymerase chain reaction" * |
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EP1618214A2 (en) * | 2003-04-25 | 2006-01-25 | Becton, Dickinson and Company | Detection of herpes simplex virus types 1 and 2 by nucleic acid amplification |
EP1618214A4 (en) * | 2003-04-25 | 2008-02-13 | Becton Dickinson Co | DETECTION OF TYPES 1 AND 2 OF HERPES SIMPLEX VIRUS BY AMPLIFICATION OF NUCLEIC ACID |
EP2071038A1 (en) * | 2003-04-25 | 2009-06-17 | Becton, Dickinson & Company | Detection of herpes simplex virus types 1 and 2 by nucleic acid amplifiction |
EP2267166A1 (en) * | 2003-04-25 | 2010-12-29 | Becton Dickinson and Company | Detection of herpes simplex virus types 1 and 2 by nucleic acid amplification |
US8221976B2 (en) | 2003-04-25 | 2012-07-17 | Becton, Dickinson And Company | Detection of herpes simplex virus types 1 and 2 by nucleic acid amplification |
EP1659187A1 (en) * | 2004-11-18 | 2006-05-24 | bioMerieux B.V. | Nucleic acid sequences that can be used as primers and probes in the amplification and detection of HSV DNA and method for the amplification and detection of HSV DNA using a transcription based amplification |
WO2006053779A3 (en) * | 2004-11-18 | 2006-08-03 | Biomerieux Bv | Nucleic acid sequences that can be used as primers and probes in the amplification and detection of hsv dna and method for the amplification and detection of hsv dna using a transcription based amplification |
US8298761B2 (en) | 2004-11-18 | 2012-10-30 | Biomerieux B.V. | Nucleic acid sequences that can be used as primers and probes in the amplification and detection of HSV DNA and method for the amplification and detection of HSV DNA using a transcription based amplification |
WO2009122201A1 (en) * | 2008-04-03 | 2009-10-08 | Genomica S.A.U. | Method for detection of herpesvirus in a test sample |
RU2470999C2 (ru) * | 2008-04-03 | 2012-12-27 | Хеномика С.А.У. | Способ детекции вируса герпеса в тестируемом образце |
Also Published As
Publication number | Publication date |
---|---|
WO1996025909A3 (es) | 1996-09-26 |
ES2093554A1 (es) | 1996-12-16 |
GB2301888A (en) | 1996-12-18 |
ES2093554B1 (es) | 1997-07-01 |
EP0789081A2 (en) | 1997-08-13 |
AU6461496A (en) | 1996-09-11 |
CA2188134A1 (en) | 1996-08-29 |
GB9621809D0 (en) | 1996-12-11 |
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