text:
|Publication number
|Publication type
|Application number
|Publication date
|Apr 29, 2008
|Filing date
|Sep 26, 2002
|Priority date
|Sep 26, 2002
|Fee status
|Also published as
|Publication number
|Thomas F. Smith, Mark J. Espy
|Original Assignee
|Mayo Foundation For Medical Education And Research
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (26), Non-Patent Citations (44), Referenced by (5), Classifications (11), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to viral diagnostics, and more particularly to detection of Epstein-Barr virus (EBV).
Epstein-Barr virus (EBV) is a member of the family Herpesviridae. EBV nucleic acid is double-stranded and consists of 172 Kb. EBV DNA is in a linear form in mature infectious virus particles and is in a circular episomal form in cells latently infected with the virus. EBV is the major cause of infectious mononucleosis, an acute, generally benign lymphoproliferative disease. Infection with EBV usually occurs by 10 years of age; 70 to 90% of children have been infected with EBV. Usually, infection in children is asymptomatic or mild and may be associated with minor illnesses such as upper respiratory tract infection, pharyngitis, tonsillitis, bronchitis, and otitis media. Other persistent EBV infections, such as those accruing in patients during the post-transplantation period, are common.
EBV has become a recognized cause of central nervous system infection and lymphoproliferative disorders (PTLD), and AIDS-related lymphomas. PTLD consists of a heterogenous group of B-cell neoplasias that arise in a setting of immunosuppression and are associated with EBV infection especially in patients lacking antibodies to this virus. The incidence of PTLD ranges from 1% for renal transplant recipients, but can be as high as 9% for heart/lung and 12% for pancreas transplant patients. EBV DNA can be detected in blood lymphocytes from patients with EBV infection, and quantitative evaluation of EBV DNA has been shown to correlate highly with the subsequent (3-4 month) development of PTLD in susceptible patients.
The invention provides for methods of identifying Epstein-Barr virus (EBV) in a biological sample. Primers and probes for detecting EBV are provided by the invention, as are kits containing such primers and probes. Methods of the invention can be used to rapidly identify EBV DNA from specimens for diagnosis of EBV infection. Using specific primers and probes, the methods of the invention include amplifying and monitoring the development of specific amplification products using fluorescence resonance energy transfer (FRET).
In one aspect of the invention, there is provided a method for detecting the presence or absence of EBV in a biological sample from an individual. The method to detect EBV includes performing at least one cycling step, which includes an amplifying step and a hybridizing step. The amplifying step includes contacting the sample with a pair of latent membrane protein (lmp) primers to produce a lmp amplification product if an EBV lmp nucleic acid molecule is present in the sample. The hybridizing step includes contacting the sample with a pair of lmp probes. Generally, the members of the pair of lmp probes hybridize within no more than five nucleotides of each other. A first lmp probe of the pair of lmp probes is typically labeled with a donor fluorescent moiety and a second lmp probe of the pair of lmp probes is labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of FRET between the donor fluorescent moiety of the first lmp probe and the acceptor fluorescent moiety of the second lmp probe. The presence of FRET is usually indicative of the presence of EBV in the sample, while the absence of FRET is usually indicative of the absence of EBV in the sample.
A pair of lmp primers generally includes a first lmp primer and a second lmp primer. A first lmp primer can include the sequence 5′-ATG AGG AAC GTG AAT CTA ATG A-3′ (SEQ ID NO:1), and a second lmp primer can include the sequence 5′-TAC AGA TAG ATG GCA CTC TTA CC-3′ (SEQ ID NO:2). A first lmp probe can include the sequence 5′-CTA CTC TCC ACG GGA TGA CTC ATC TCA ACA CA-3′ (SEQ ID NO:3), and a second lmp probe can include the sequence 5′-GAA GAA GCG GGC AGA GG-3′ (SEQ ID NO:4).
In some aspects, one of the lmp primers can be labeled with a fluorescent moiety (either a donor or acceptor, as appropriate) and can take the place of one of the lmp probes.
The members of the pair of lmp probes can hybridize within no more than two nucleotides of each other, or can hybridize within no more than one nucleotide of each other. A representative donor fluorescent moiety is fluorescein, and corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Additional corresponding donor and acceptor fluorescent moieties are known in the art.
In one aspect, the detecting step includes exciting the sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the acceptor fluorescent moiety (i.e., visualizing and/or measuring FRET). In another aspect, the detecting step includes quantitating the FRET. In yet another aspect, the detecting step can be performed after each cycling step (e.g., in real-time).
Generally, the presence of FRET within 45 cycles (e.g., 20, 25, 30, 35, or 40 cycles) indicates the presence of an EBV infection in the individual. In addition, determining the melting temperature between one or both of the lmp probe(s) and the lmp amplification product can confirm the presence or absence of the EBV.
Representative biological sample include dermal swabs, lymphoid tissue, cerebrospinal fluid, blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine. The above-described methods can further include preventing amplification of a contaminant nucleic acid. Preventing amplification can include performing the amplifying step in the presence of uracil and treating the sample with uracil-DNA glycosylase prior to amplifying.
In addition, the cycling step can be performed on a control sample. A control sample can include the same portion of the EBV lmp nucleic acid molecule. Alternatively, a control sample can include a nucleic acid molecule other than an EBV lmp nucleic acid molecule. Cycling steps can be performed on such a control sample using a pair of control primers and a pair of control probes. The control primers and probes are other than lmp primers and probes. One or more amplifying steps produces a control amplification product. Each of the control probes hybridizes to the control amplification product.
In another aspect of the invention, there are provided articles of manufacture, or kits. Kits of the invention can include a pair of lmp primers, and a pair of lmp probes, and a donor and corresponding acceptor fluorescent moieties. For example, the first lmp primer provided in a kit of the invention can have the sequence 5′-ATG AGG AAC GTG AAT CTA ATG A-3′ (SEQ ID NO:1) and the second lmp primer can have the sequence 5′-TAC AGA TAG ATG GCA CTC TTA CC-3′ (SEQ ID NO:2). The first lmp probe provided in a kit of the invention can have the sequence 5′-CTA CTC TCC ACG GGA TGA CTC ATC TCA ACA CA-3′ (SEQ ID NO:3) and the second lmp probe can have the sequence 5′-GAA GAA GCG GGC AGA GG-3′ (SEQ ID NO:4).
Articles of manufacture can include fluorophoric moieties for labeling the probes or the probes can be already labeled with donor and corresponding acceptor fluorescent moieties. The article of manufacture can also include a package insert having instructions thereon for using the primers, probes, and fluorophoric moieties to detect the presence or absence of EBV in a sample.
In yet another aspect of the invention, there is provided a method for detecting the presence or absence of EBV in a biological sample from an individual. Such a method includes performing at least one cycling step. A cycling step can include an amplifying step and a hybridizing step. Generally, an amplifying step includes contacting the sample with a pair of lmp primers to produce a lmp amplification product if an EBV lmp nucleic acid molecule is present in the sample. Generally, a hybridizing step includes contacting the sample with a lmp probe. Such a lmp probe is usually labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the lmp probe. The presence or absence of fluorescence is indicative of the presence or absence of EBV in said sample.
In one aspect, amplification can employ a polymerase enzyme having 5′ to 3′ exonuclease activity. Thus, the first and second fluorescent moieties would be within no more than 5 nucleotides of each other along the length of the probe. In another aspect, the lmp probe includes a nucleic acid sequence that permits secondary structure formation. Such secondary structure formation generally results in spatial proximity between the first and second fluorescent moiety. According to this method, the second fluorescent moiety on a probe can be a quencher.
In another aspect of the invention, there is provided a method for detecting the presence or absence of EBV in a biological sample from an individual. Such a method includes performing at least one cycling step. A cycling step can include an amplifying step and a dye-binding step. An amplifying step generally includes contacting the sample with a pair of lmp primers to produce a lmp amplification product if an EBV lmp nucleic acid molecule is present in the sample. A dye-binding step generally includes contacting the lmp amplification product with a double-stranded DNA binding dye. The method further includes detecting the presence or absence of binding of the double-stranded DNA binding dye into the amplification product. According to the invention, the presence of binding is typically indicative of the presence of EBV in the sample, and the absence of binding is typically indicative of the absence of EBV in the sample. Such a method can further include the steps of determining the melting temperature between the lmp amplification product and the double-stranded DNA binding dye. Generally, the melting temperature confirms the presence or absence of EBV. Representative double-stranded DNA binding dyes include SYBRGreenI®, SYBRGold®, and ethidium bromide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.
A real-time assay for qualitatively and quantitatively detecting EBV in a biological sample that is more sensitive and specific than existing assays is described herein. The assay detects EBV in cerebrospinal fluid (CSF) of patients with central nervous system (CNS) disease. The assay also detects EBV in blood or plasma specimens from patients with viremia. The invention provides primers and probes for detecting EBV infections and articles of manufacture containing such primers and probes. The increased sensitivity of real-time PCR for detection of EBV compared to other methods, as well as the improved features of real-time PCR including sample containment and real-time detection of the amplified product, make feasible the implementation of this technology for routine diagnosis of EBV infections in the clinical laboratory.
EBV Nucleic Acids and Oligonucleotides
The invention provides methods to detect EBV by amplifying, for example, a portion of the EBV latent membrane protein (lmp) nucleic acid. EBV nucleic acids other than those exemplified herein (e.g., other than lmp) also can be used to detect EBV in a sample and are known to those of skill in the art. The nucleic acid sequence of EBV lmp is available (see, for example, GenBank Accession No. X81759). Specifically, primers and probes to amplify and detect EBV lmp nucleic acid molecules are provided by the invention.
Primers that amplify an EBV nucleic acid molecule, e.g., EBV lmp, can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights, Inc., Cascade, Colo.). Important features when designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (i.e., the primers need to be long enough to anneal with sequence-specificity and to initiate synthesis but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 15 to 30 nucleotides in length. As used herein, "lmp primers" refers to oligonucleotide primers that anneal specifically to lmp nucleic acid sequences and initiate synthesis therefrom under appropriate conditions.
Designing oligonucleotides to be used as hybridization probes can be performed in a manner similar to the design of primers, although the members of a pair of probes preferably anneal to an amplification product within no more than 5 nucleotides of each other on the same strand such that FRET can occur (e.g., within no more than 1, 2, 3, or 4 nucleotides of each other). This minimal degree of separation typically brings the respective fluorescent moieties into sufficient proximity such that FRET occurs. It is to be understood, however, that other separation distances (e.g., 6 or more nucleotides) are possible provided the fluorescent moieties are appropriately positioned relative to each other (for example, with a linker arm) such that FRET can occur. In addition, probes can be designed to hybridize to targets that contain a polymorphism or mutation, thereby allowing differential detection of EBV strains or mutants based on either absolute hybridization of different pairs of probes corresponding to the particular EBV strain or mutant to be distinguished or differential melting temperatures between, for example, members of a pair of probes and each amplification product corresponding to a EBV strain or mutant to be distinguished. As with oligonucleotide primers, oligonucleotide probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15 to 30 nucleotides in length. As used herein, "lmp probes" refers to oligonucleotide probes that specifically anneal to lmp amplification products.
Constructs of the invention include vectors containing an EBV nucleic acid molecule, e.g., EBV lmp or a fragment thereof. Constructs of the invention can be used, for example, as control template nucleic acid molecules. Vectors suitable for use in the present invention are commercially available and/or produced by recombinant DNA technology methods routine in the art. EBV lmp nucleic acid molecules can be obtained, for example, by chemical synthesis, direct cloning from EBV, or by PCR amplification. An EBV nucleic acid molecule or fragment thereof can be operably linked to a promoter or other regulatory element such as an enhancer sequence, a response element, or an inducible element that modulates expression of the EBV nucleic acid molecule. As used herein, operably linking refers to connecting a promoter and/or other regulatory elements to an EBV nucleic acid molecule in such a way as to permit and/or regulate expression of the EBV nucleic acid molecule. A promoter that does not normally direct expression of EBV lmp can be used to direct transcription of a lmp nucleic acid using, for example, a viral polymerase, a bacterial polymerase, or a eukaryotic RNA polymerase II. Alternatively, the lmp native promoter can be used to direct transcription of a lmp nucleic acid, respectively, using, for example, an RNA polymerase enzyme (e.g., RNA polymerase II). In addition, operably linked can refer to an appropriate connection between a EBV lmp promoter or regulatory element and a heterologous coding sequence (i.e., a non-lmp coding sequence, for example, a reporter gene) in such a way as to permit expression of the heterologous coding sequence.
Constructs suitable for use in the methods of the invention typically include, in addition to EBV lmp nucleic acid molecules, sequences encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting desired constructs and/or transformants, and an origin of replication. The choice of vector systems usually depends upon several factors, including, but not limited to, the choice of host cells, replication efficiency, selectability, inducibility, and the ease of recovery.
Constructs of the invention containing EBV lmp nucleic acid molecules can be propagated in a host cell. As used herein, the term host cell is meant to include prokaryotes and eukaryotes. Prokaryotic hosts may include E. coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. A construct of the invention can be introduced into a host cell using any of the techniques commonly known to those of ordinary skill in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be delivered directly to cells (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).
Polymerase Chain Reaction (PCR)
U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in the present invention include oligonucleotide primers capable of acting as a point of initiation of nucleic acid synthesis within EBV lmp sequences. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating.
The term "thermostable polymerase" refers to a polymerase enzyme that is heat stable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3′ end of each primer and proceeds in the 5′ to 3′ direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished.
If the EBV template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90° C. to about 105° C. for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 30 sec to 4 min.
If the double-stranded nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence on the EBV nucleic acid. The temperature for annealing is usually from about 35° C. to about 65° C. Annealing times can be from about 10 secs to about 1 min. The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer such that products complementary to the template nucleic acid are generated. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40° to 80° C.). Extension times can be from about 10 secs to about 5 mins.
PCR assays can employ EBV nucleic acid such as DNA or RNA, including messenger RNA (mRNA). The template nucleic acid need not be purified; it may be a minor fraction of a complex mixture, such as EBV nucleic acid contained in human cells. DNA or RNA may be extracted from a biological sample such as dermal swabs, lymphoid tissue, cerebrospinal fluid, blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington D.C.). Nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.
The oligonucleotide primers are combined with PCR reagents under reaction conditions that induce primer extension. For example, extension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured template DNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.
The newly synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the target EBV nucleic acid molecule. The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.
Fluorescence Resonance Energy Transfer (FRET)
FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on a concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent moieties that can be visualized or otherwise detected and/or quantitated. Two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to an amplification product at particular positions determined by the complementarity of the oligonucleotide probes to the target nucleic acid sequence. Upon hybridization of the oligonucleotide probes to the amplification product at the appropriate positions, a FRET signal is generated. Hybridization temperatures can range from about 35° C. to about 65° C. for about 10 sees to about 1 min.
Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system or a fluorometer. Excitation to initiate energy transfer can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range.
As used herein with respect to donor and corresponding acceptor fluorescent moieties "corresponding" refers to an acceptor fluorescent moiety having an emission spectrum that overlaps the excitation spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Accordingly, efficient non-radiative energy transfer can be produced therebetween.
Fluorescent donor and corresponding acceptor moieties are generally chosen for (a) high efficiency Förster energy transfer; (b) a large final Stokes shift (>100 nm); (c) shift of the emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) shift of the emission to a higher wavelength than the Raman water fluorescent emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be chosen that has its excitation maximum near a laser line (for example, Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, a high quantum yield, and a good overlap of its fluorescent emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be chosen that has a high extinction coefficient, a high quantum yield, a good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red part of the visible spectrum (>600 nm).
Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid, 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives. Representative acceptor fluorescent moieties, depending upon the donor fluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of Lanthanide ions (e.g., Europium, or Terbium). Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).
The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important, as the linker arms will affect the distance between the donor and acceptor fluorescent moieties. The length of a linker arm for the purpose of the present invention is the distance in Angstroms (Å) from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 to about 25 Å. The linker arm may be of the kind described in WO 84/03285. WO 84/03285 also discloses methods for attaching linker arms to a particular nucleotide base, and also for attaching fluorescent moieties to a linker arm.
An acceptor fluorescent moiety such as an LC™-Red 640-NHS-ester can be combined with C6-Phosphoramidites (available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LC™-Red 640-Phosphoramidite. Frequently used linkers to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, for example, fluorescein-CPG's from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis.
Detection of EBV
Serological assays for EBV have been used to clinically detect EBV (see, for example, Klein, 1976, Bull. Cancer, 63:399-410). In recent years, PCR detection of EBV in patients diagnosed with lymphomas of the CNS has replaced cell culture techniques that have been ineffective for detecting herpesvirus species from CSF.
Several amplification assays have been reported for the qualitative and quantitative detection of EBV. These PCR assays require the use of conventional methods such as gel electrophoresis and Southern blotting for detection of amplified EBV DNA. These methods are labor intensive and frequently require more than one day to complete. Additionally, the manipulation of amplification products for the purpose of detection (e.g., by blotting) increases the risk of carry-over contamination and false positives. Further disadvantages of current PCR methods for detecting EBV include poor standardization of reagents and product amplification conditions, and the lack of general availability for use in all diagnostic and clinical laboratories.
The invention provides a qualitative and quantitative assay for detecting EBV. Identical PCR cycling conditions are used for both the qualitative and quantitative assays. The quantitative levels of EBV DNA are determined by processing individual samples containing known amounts of target EBV nucleic acid as standards along with samples from patients. The LightCycler PCR assay is the first automated, real-time system for the qualitative and quantitative detection of EBV DNA. The system is rapid (2-3 hours total sample preparation and analytical time), is sensitive (detects ≧10 copies of EBV DNA/sample), specific (detects EBV DNA target exclusively), and has a wide dynamic linear range of 101 to 107 copies of EBV DNA/sample. By using commercially available real-time PCR instrumentation (e.g. LightCycler™, Roche Molecular Biochemicals, Indianapolis, Ind.), PCR amplification and detection of the amplification product can be combined in a single closed cuvette with dramatically reduced cycling time. Since detection occurs concurrently with amplification, the real-time PCR methods obviate the need for manipulation of the amplification product, and diminish the risk of cross-contamination between amplification products. Real-time PCR greatly reduces turn-around time and is an attractive alternative to conventional PCR techniques in the clinical laboratory.
The present invention provides methods for detecting the presence or absence of EBV in a biological sample from an individual. Methods provided by the invention avoid problems of sample contamination, false negatives, and false positives. The methods include performing at least one cycling step that includes amplifying an EBV portion of a lmp nucleic acid molecule from a sample using a pair of lmp primers, respectively. Each of the lmp primers anneals to a target within or adjacent to an EBV lmp nucleic acid molecule such that at least a portion of each amplification product contains nucleic acid sequence corresponding to lmp. More importantly, the amplification product should contain the nucleic acid sequences that are complementary to the lmp probes. The lmp amplification product is produced provided that EBV nucleic acid is present. Each cycling step further includes contacting the sample with a pair of lmp probes. According to the invention, one member of each pair of the lmp probes is labeled with a donor fluorescent moiety and the other is labeled with a corresponding acceptor fluorescent moiety. The presence or absence of FRET between the donor fluorescent moiety of the first lmp probe and the corresponding acceptor fluorescent moiety of the second lmp probe is detected upon hybridization of the lmp probes to the lmp amplification product.
Each cycling step includes an amplification step and a hybridization step, and each cycling step is usually followed by a FRET detecting step. Multiple cycling steps are performed, preferably in a thermocycler. Methods of the invention can be performed using the lmp primer and probe sets to detect the presence of EBV.
As used herein, "amplifying" refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid molecule (e.g., EBV lmp nucleic acid molecules). Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme (e.g., MgCl2 and/or KCl).
If amplification of EBV nucleic acid occurs and an amplification product is produced, the step of hybridizing results in a detectable signal based upon FRET between the members of the pair of probes. As used herein, "hybridizing" refers to the annealing of probes to an amplification product. Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.
Generally, the presence of FRET indicates the presence of EBV in the sample, and the absence of FRET indicates the absence of EBV in the sample. Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and/or accuracy of a test result, however. Using the methods disclosed herein, detection of FRET within 45 cycling steps is indicative of an EBV infection.
Methods of the invention also can be used for EBV vaccine efficacy studies or epidemiology studies. For example, an attenuated EBV in a vaccine can be detected using the methods of the invention during the time when the virus is still present in an individual. For such vaccine efficacy studies, the methods of the invention can be used to determine, for example, the persistence of an attenuated strain of EBV used in a vaccine, or can be performed in conjunction with an additional assay such as a serologic assay to monitor an individual's immune response to such a vaccine. In addition, methods of the invention can be used to distinguish one EBV strain from another for epidemiology studies of, for example, the origin or severity of an outbreak of EBV.
Representative biological samples that can be used in practicing the methods of the invention include dermal swabs, lymphoid tissue, cerebrospinal fluid, blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, and urine. Collection and storage methods of biological samples are known to those of skill in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and/or kits known in the art) to release EBV nucleic acid or in some cases, the biological sample can be contacted directly with the PCR reaction components and the appropriate oligonucleotides.
Melting curve analysis is an additional step that can be included in a cycling profile. Melting curve analysis is based on the fact that a nucleic acid sequence melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA duplexes have separated into single strands. The melting temperature of a DNA depends primarily upon its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than those having an abundance of A and T nucleotides. By detecting the temperature at which the FRET signal is lost, the melting temperature of probes can be determined. Similarly, by detecting the temperature at which signal is generated, the annealing temperature of probes can be determined. The melting temperature(s) of the lmp probes from the respective amplification product can confirm the presence or absence of EBV in the sample.
Within each thermocycler run, control samples are cycled as well. Positive control samples can amplify EBV nucleic acid control template (e.g., a nucleic acid other than lmp) using, for example, control primers and control probes. Positive control samples can also amplify, for example, a plasmid construct containing an EBV lmp nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run side-by-side with the patients' samples. Each thermocycler run should also include a negative control that, for example, lacks EBV template DNA. Such controls are indicators of the success or failure of the amplification, hybridization and/or FRET reaction. Therefore, control reactions can readily determine, for example, the ability of primers to anneal with sequence-specificity and to initiate elongation, as well as the ability of probes to hybridize with sequence-specificity and for FRET to occur.
In an embodiment, the methods of the invention include steps to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next. In addition, standard laboratory containment practices and procedures are desirable when performing methods of the invention. Containment practices and procedures include, but are not limited to, separate work areas for different steps of a method, containment hoods, barrier filter pipette tips and dedicated air displacement pipettes. Consistent containment practices and procedures by personnel are necessary for accuracy in a diagnostic laboratory handling clinical samples.
Conventional PCR methods in conjunction with FRET technology can be used to practice the methods of the invention. In one embodiment, a LightCycler™ instrument is used. A detailed description of the LightCycler™ System and real-time and on-line monitoring of PCR can be found at biochem.roche.com/lightcycler on the World Wide Web The following patent applications describe real-time PCR as used in the LightCycler™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™ instrument is a rapid thermal cycler combined with a microvolume fluorometer utilizing high quality optics. This rapid thermocycling technique uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber are controlled by alternating heated and ambient air. Due to the low mass of air and the high ratio of surface area to volume of the cuvettes, very rapid temperature exchange rates can be achieved within the LightCycler™ thermal chamber. Addition of selected fluorescent dyes to the reaction components allows the PCR to be monitored in real time and on-line. Furthermore, the cuvettes serve as an optical element for signal collection (similar to glass fiber optics), concentrating the signal at the tip of the cuvette. The effect is efficient illumination and fluorescent monitoring of microvolume samples.
The LightCycler™ carousel that houses the cuvettes can be removed from the instrument. Therefore, samples can be loaded outside of the instrument (in a PCR Clean Room, for example). In addition, this feature allows for the sample carousel to be easily cleaned and sterilized. The fluorometer, as part of the LightCycler™ apparatus, houses the light source. The emitted light is filtered and focused by an epi-illumination lens onto the top of the cuvette. Fluorescent light emitted from the sample is then focused by the same lens, passed through a dichroic mirror, filtered appropriately, and focused onto data-collecting photohybrids. The optical unit currently available in the LightCycler™ instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468) includes three band-pass filters (530 nm, 640 nm, and 710 nm), providing three-color detection and several fluorescence acquisition options. Data collection options include once per cycling step monitoring, fully continuous single-sample acquisition for melting curve analysis, continuous sampling (in which sampling frequency is dependent on sample number) and/or stepwise measurement of all samples after defined temperature interval.
The LightCycler™ can be operated using a PC workstation and can utilize a Windows NT operating system. Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit. The software can display the fluorescence signals in real-time immediately after each measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence vs. cycle number can be continually updated for all samples. The data generated can be stored for further analysis.
As an alternative to FRET, an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBRGreenI® or SYBRGold® (Molecular Probes)). Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A double-stranded DNA binding dye such as a nucleic acid intercalating dye also can be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product.
As described herein, amplification products also can be detected using labeled hybridization probes that take advantage of FRET technology. A common format of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and are generally designed to hybridize in close proximity to each other in a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, for example, fluorescein, is excited at 470 nm by the light source of the LightCycler™ Instrument. During FRET, the fluorescein transfers its energy to an acceptor fluorescent moiety such as LightCycler®-Red 640 (LC™-Red 640) or LightCycler™-Red 705 (LC™-Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler™ instrument. Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of EBV genomes).
Another FRET format utilizes TaqMan® technology to detect the presence or absence of an amplification product, and hence, the presence or absence of EBV. TaqMan® technology utilizes one single-stranded hybridization probe labeled with two fluorescent moieties. When a first fluorescent moietyis excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded by the 5′ to 3′ exonuclease activity of the Taq Polymerase during the subsequent elongation phase. As a result, the excited fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected. By way of example, an ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) uses TaqMan® technology, and is suitable for performing the methods described herein for detecting EBV. Information on PCR amplification and detection using an ABI PRISM® 770 system can be found at appliedbiosystems.com/products on the World Wide Web.
Molecular beacons in conjunction with FRET also can be used to detect the presence of an amplification product using the real-time PCR methods of the invention. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g., a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the target nucleic acids (i.e., amplification products), the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.
It is understood that the present invention is not limited by the configuration of one or more commercially available instruments.
Articles of Manufacture
The invention further provides for articles of manufacture to detect EBV. An article of manufacture according to the present invention can include primers and probes used to detect EBV, together with suitable packaging materials. Representative primers and probes for detection of EBV are capable of hybridizing to EBV lmp nucleic acid molecules. Methods of designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to EBV lmp nucleic acid molecules are provided.
Articles of manufacture of the invention also can include one or more fluorescent moieties for labeling the probes or, alternatively, the probes supplied with the kit can be labeled. For example, an article of manufacture may include a donor fluorescent moiety for labeling one of the lmp probes and an acceptor fluorescent moiety for labeling the other lmp probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.
Articles of manufacture of the invention also can contain a package insert or package label having instructions thereon for using the lmp primers and probes to detect EBV in a sample. Articles of manufacture may additionally include reagents for carrying out the methods disclosed herein (e.g., buffers, polymerase enzymes, co-factors, or agents to prevent contamination). Such reagents may be specific for one of the commercially available instruments described herein.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Oligonucleotide Primers and Probes
Primers and probes were designed using the OLIGO software (Molecular Biology Insights, Inc., Cascade, OR). Primers were synthesized on a 0.2 μM scale by the Mayo Molecular Biology Core Facility (Rochester, MN). Probes were obtained from Idaho Technologies (idahotech.com/itbiochem/index on the World Wide Web). Sequences for primers and probes are shown in Table 1. The GenBank Accession numbers for the reference sequences used to design the primers and probes for each target are shown in Table 1, along with the relative location of each primer and probe.
Primers and Probes for the Detection of EBV
Gene Target
Size, bp)
Probe Name
5′-ATG AGG AAC GTG AAT CTA ATG A-3′ (Forward Primer)
protein (lmp)
5′-TAC AGA TAG ATG GCA CTC TTA CC-3′ (Reverse Primer)
Primers were adjusted to 50 μM by measuring the A260 of a 1/50 dilution (196 μl water+4 μl, Dilution Factor (DF)=50). The concentration was estimated by the following formula:
(μM found/50)×μl remaining)−μl remaining=water to add
Probes were dissolved in TE′ to a concentration of 20 μM (supplied with the probes and resuspended according to manufacturer's instructions). The concentration of oligonucleotides and dye was double checked by UV absorption using the following equations from Biochemica, 1999,1:5-8:
Abs max
Max (nm)
LC Red 640
Plasmid controls were produced by cloning the lmp product amplified by the lmp primers into the 2.1 TOPO TA cloning vector (Invitrogen Corp., Carlsbad, Calif.). The recombinant vectors were transformed into chemically competent E. coli cells. The correct recombinant plasmid was confirmed and purified with a Wizard MiniPrep (Promega Corp., Madison, Wis.) Cleaning kit. The stock concentrations of the controls (in genomic equivalents) were determined. The plasmid containing the lmp insert was used to determine the analytical sensitivity of the assay. Plasmid concentration or the copy number of the gene target insert was determined with the following formula:
PCR Conditions
Nucleic acids are extracted from 0.2 ml of cerebrospinal fluid (qualitative) or 1.0 ml anticoagulated (EDTA) blood (quantitative) from patients using the MagNA Pure instrument (Roche Diagnostics, Indianapolis, Ind.).
For the assay, a 5 μl aliquot of extracted nucleic acid was added to 15 μl of PCR reaction mixture in each reaction capillary. A no-target control received 15 μl of reaction mixture with 5 μl water.
LightCycler ™TM Master Mix - EBV lmp
10 X buffer
Platinum ® Taq
dNTP Plus
Total volume
The PCR reagents and specimen extract are centrifuged in the capillary to facilitate mixing. All capillaries are then sealed and amplified using the following protocol.
Fluorescence Settings:
LED Power
F1 Gain
F2 Gain
Quantification Settings:
Channel Settings
Name of Program
Experimental Protocol:
The lmp primers and probes were used for both qualitative and quantitative EBV detection. Amplification of the EBV lmp sequences using the lmp primers disclosed herein results in a 238 bp amplification product.
Quantitative Analysis of EBV
For quantitative analysis, ten-fold serial dilutions of the target gene plasmid were prepared to yield standardized levels of EBV lmp DNA ranging from 101 up to 1×1010. The serial dilutions were processed in the LightCycler instrument along with DNA extracted from a patient's EDTA-anticoagulated specimen. A curve plotting the cycle number (crossover point) versus the log of the concentration of DNA in each serial dilution was produced. The quantitative level of EBV DNA in patient sample was determined by comparison of the crossover point from the three standard dilutions of the target.
Melting Curves
Following the completion of the amplification reaction, a melting curve analysis was performed by raising the temperature in the LightCycler™ thermal chamber from 50° C. to 85° C. at 0.2° C. per second. Fluorescent measurements were taken continuously as the temperature was raised and a melting curve was generated. The pair of lmp probes had a specific and characteristic melting curve from the lmp amplification product. The melting temperature (Tm) for the lmp product generated was about 62° C.
Analytical Sensitivity
Serial dilutions of the EBV plasmid controls described in Example 3 were used to examine the sensitivity of the EBV LightCycler™ assay. The analytical sensitivity of the EBV LightCycler™ assay was at least 10 copies of the target sequence.
Analytical Specificity
The following members of the Herpesvirus family were used to examine the specificity of the EBV LightCycler™ assay: HSV-1, HSV-2, VZV, CMV, HHV-6, HHV-7, HHV-8. In addition, nucleic acid from S. aureus, S. epidermidis, E. coli, and human cells was isolated and used to examine the specificity of the EBV LightCycler assay. The lmp primers and probes under the amplification conditions described herein did not cross-react with nucleic acid from any of the above-listed organisms.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
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Owner name: MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH
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Topics: law-crime-and-justice, unrest-conflict-and-war, afghanistan, england
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Jim Maxwell from ABC Grandstand provides analysis on the Australia-India Test at the Sydney Cricket Ground.
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The asylum seeker debate increasingly resembles a dialogue between characters in a parallel world beyond the looking glass.
The crucial issues are confidently mis-identified and then policy responses to resolve them are stridently asserted.
However, the reality is more nuanced and straightforward than the public discourse would suggest.
Topics: refugees, immigration, government-and-politics, community-and-society
The Gillard Government is rather fond of reminding us that it is committed to giving more Australians access to a university education, but so far it has just been hollow spin.
Topics: university-and-further-education, government-and-politics, federal-government, education
Libertarianism is like pure communism and pure capitalism. A nice thought experiment for ivory tower types.
Topics: government-and-politics, world-politics
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Topics: maritime, police, point-piper-2027
ABC Weather
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Jewish settlers are being blamed for a rising number of hate crimes against Palestinians in the West Bank.
Topics: race-relations, unrest-conflict-and-war, world-politics, judaism, islam, israel, palestinian-territory-occupied

subset: commoncrawl


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Princess Grace and PoppyI Can Read/Princess Parables
By Jeanna Young Jacqueline Johnson
ZONDERVANCopyright © 2012 Jeanna Young
All right reserved.
Chapter OnePrincess Grace lives in a castle. She has four sisters. They are Joy, Faith, Charity, and Hope. Their daddy is the king!
One day, Grace dropped a vase. While she was picking up flowers, Grace heard a noise. "Meow, meow, meow!"
Excerpted from Princess Grace and Poppy by Jeanna Young Jacqueline Johnson Copyright © 2012 by Jeanna Young. Excerpted by permission of ZONDERVAN. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.


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Normalization of slant and skew is often used in processing a word image before recognition. In this paper, we prove the theoretical equivalence of different methods for slant and skew corrections. In particular, we show that correcting first for skew by rotation and then for slant by a shear transformation in the horizontal direction is equivalent to first correcting for slant by a shear transformation in the horizontal direction and then for skew by a shear transformation in the vertical direction. Our proof can be easily modified to prove equivalence of other methods for correcting the slant and skew
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Apply for it.Amenity and Arts Grant Scheme 2016 Resident Association Grant Scheme 2016 Planning Permission, Housing, Jobs, Register to Vote Education Grants Event in a Public Place Tenders Residents Parking Permit Licence to place and maintain an appliance on, over or along a public road Scaffolding and Hoarding Application form Table & Chairs licence application form Vacant Property Grant Incentive Scheme more...
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The Future of North America
Replacing a Bad Neighbor Policy
On January 20, 2009, if not before, a new national security adviser will tell the incoming president of the United States that the first two international visitors should be the prime minister of Canada and the president of Mexico. Almost every new president since World War II has followed this ritual, because no two countries in the world have a greater impact economically, socially, and politically on the United States than its neighbors. The importance of Canada and Mexico may, however, come as a surprise to most Americans, as well as to the new president. In the presidential campaign, instead of discussing a positive agenda for North America's future, the candidates have focused critically on two parts of that agenda, the 14-year-old North American Free Trade Agreement (NAFTA) and immigration. And overall, one could conclude from listening to the campaign that Iraq is key to U.S. national security, China is the United States' most important trading partner, and Saudi Arabia and Venezuela supply most of the United States' energy.
None of these propositions is true. For most of the past decade, Canada and Mexico have been the United States' most important trading partners and largest sources of energy imports. U.S. national security depends more on cooperative neighbors and secure borders than it does on defeating militias in Basra.
The new president will take office at a low moment in U.S. relations with its neighbors. The percentage of Canadians and Mexicans who have a favorable view of U.S. policy
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|Publication number
|Publication type
|Application number
|Publication date
|Mar 23, 1993
|Filing date
|May 15, 1991
|Priority date
|May 15, 1991
|Fee status
|Publication number
|Pieter W. C. J. le Blanc, Timothy R. Maher
|Original Assignee
|Nimbus, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (6), Referenced by (28), Classifications (13), Legal Events (10)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to a mouse-like computer accessory which enables visually handicapped operators to read text on a computer screen in Braille format, and more particularly to a novel Braille displaying mechanism for such a device.
Various method have been proposed for translating text on a computer screen into Braille symbols capable of being perceived by visually handicapped persons. Among such methods is the use of a mouse which can be used to move a cursor along text lines on a screen, and which contains a set of raisable pins that spell out in Braille the character at the cursor position. A problem with such mice is that they require a plurality of solenoids (usually six) to operate the Braille pins. As a result, such devices are cumbersome and expensive, consume substantial power, generate considerable heat in prolonged use, and present maintenance problems. Consequently, there is a need for a simple, inexpensive, compact and reliable mechanism for actuating the pins of a Braille mouse.
The invention fills the above-stated need by providing a movable character element bearing character indicia which, when pressed into engagement with appropriately configured Braille pins, cause a selected pin combination to be raised above the surface of the device under the control of a single solenoid. In one preferred embodiment of the invention, the character element may take the shape of a character disc with axial flexibility but radial integrity, using a coded wheel mechanism to circumferentially position the rim of the disc under the Braille pins, and using a single solenoid to push the disc into engagement with the pin.
In another preferred embodiment, the character element is a flexible endless belt carrying the character indicia. The single solenoid presses a selected section of the belt into engagement with the Braille pins.
In a third preferred embodiment of the invention, the character element is a hard wheel which carries the character indicia on its rim. The wheel is normally spring-biased against the Braille pins but is retracted away from them by the single solenoid while the wheel is rotated to another character position.
In a second aspect of the invention common to all three preferred embodiments, the size of the character element is minimized without loss of characters by encoding the character indicia in a linear pattern, and translating that pattern into the box pattern of the Braille alphabet by using eccentrically actuated push pins or fingers.
FIG. 1 is a perspective outside view of the device of this invention;
FIG. 2 is a vertical section through a first embodiment of the device of FIG. 1 illustrating a disc-type actuating mechanism in accordance with this invention;
FIG. 3 is a generalized plan view of the character disc of FIG. 2;
FIG. 3A is a detail plan view of a portion of the character disc;
FIG. B is a detail sction along line 3B,C--3B,C of FIG. 3A illustrating a sub-embodiment in which the character indicia raise all except selected pins;
FIG. 3C is a detail section along line 3B,C--3B,C of FIG. 3A illustrating another sub-embodiment in which the character indicia raise only selected pins;
FIG. 4 is a perspective view of a push pin used in the mouse of FIG. 1;
FIG. 5 is an enlarged partial plan view of the Braille pin pad;
FIG. 6 is an enlarged assembled vertical section of the pin mechanism of FIG. 1;
FIG. 6A is an exploded detail view of the pin mechanism;
FIG. 7 is a side elevational view of a second embodiment illustrating a belt-type actuating mechanism;
FIG. 8 is an end elevational view of the embodiment of FIG. 7;
FIG. 8A is a section along line 8A of FIG. 7;
FIG. 9 is a side elevational view of a third embodiment illustrating a wheel-type actuating mechanism; and
FIG. 10 is an end elevational view of the embodiment of FIG. 9.
In the following discussion, the device of this invention is described as a mouse, i.e. a device which conventionally uses a track ball to electronically move a cursor on a computer screen. It should be understood, however, that the invention may equally be embodied, with the same advantages, in a stationary mouse-like accessory used in connjunction with a keyboard or other apparatus which allows moving the cursor position independently of the inventive device.
FIG. 1 illustrates the Braille mouse 10 of this invention from the user's perspective. Preferably, the mouse 10 is grasped between the thumb and little finger, with the index finger resting on the line-up button 16, the middle finger resting on the Braille pad 14, and the ring finger resting on the line-down button 12 (as explained below, the mouse 10 can move the cursor only horizontally). As the mouse 10 is moved from side to side to scan a line of text on a computer screen (not shown), the pins 18 on the Braille pad 14 at any given time spell out the character which appears at the cursor location at that time.
A first preferred mechanism which accomplishes the foregoing objective is shown in FIG. 2. The movement of the mouse 10 is conventionally sensed by the track ball 20 and is translated by conventional sensor electronics 22 into cursor positioning coordinate changes which are transmitted to the computer. To prevent unintended line changes, the vertical motion sensor in electronics 22 is physically or electronically disables in the mouse 10 when the mouse 10 is used as a Braille mouse. In that mode, buttons 12 and 16 are used to convey vertical positioning information to the computer. Alternatively, in a track ball-less embodiment of the invention, the cursor position coordinates may be changed by, e.g., keystrokes on a keyboard.
The identity of the character at the cursor position is conveyed to the mouse 10 by conventionally designed electronics in the computer (not shown), whose nature depends upon the type of computer involved and is not material to this invention. This identity information is translated by appropriate electronics 24 into an actuating signal for the stepper motor 26 which turns the disc spindle 28. Mounted on the spindle 28 is a code information disc 30 whose rim 32 can be pushed into engagement with the pins 18 by the actuator arm 34 upon energization of the solenoid 36. Conventional interlock electronics 38 assure that the solenoid 36 is de-energized while the disc 30 is in motion.
The code information disc 30 is shown in more detail in FIGS. 3 and 3A. The disc 30 must satisfy two sets of inconsistent requirements: it must be radially stiff and strong but axially highly flexible, and it must be small yet carry on its rim 32 all sixty-four possible combinations of the six Braille pins 18 (the sixty-four character set can be expanded to 126 characters or more by using one or more of the characters as a prefix which appears momentarily before another character).
To satisfy the former requirement set, the disc 30 is provided with cutouts 40 between its hub 42 and its character code carrying rim 32. The cutouts 40, the thinness of the web 44 between the hub 42 and the rim 32, and the radial grooves 46 formed between the character pads 48 all cooperate to give the disc 30 considerable flexibility in an axial direction while preserving its radial and circumferential integrity. An index marker 49 may be provided to conventionally cooperate with an optical, magnetic or mechanical indexing device (not shown) to maintain the positional accuracy of the character disc 30.
The second requirement set is satisfied by arranging the characer code for each characer in a straight line rather than in the box format of Braille symbols. Specifically, each character pad 48 carries a character code in the form of indicia 50 (openings 50a or depressions 50b in the rim surface in FIG. 3B) whose radial positions "1" through "6" correspond to the numbers of the Braille pins 18 which remain retracted when that character is displayed.
Alternatively, the indicia 50 may be protuberances 50c (FIG. 3C) whose radial positions "1" through "6" correspond to the numbers of the Braille pins 18 which are to be extended.
The radially linear arrangement of the character codes on the character pads 48 is translated into the Braille-cell form by the eccentric push pins 18 which are shown in more detail in FIGS. 4 and 5. Each pin 18 has a flange 52 from which an eccentrically mounted finger 54 extends downwardly. As indicated in FIG. 5, the eccentricities of the fingers 54 are so oriented that the fingers 54 lie in a radial line with respect to the disc hub 42.
FIGS. 6 and 6A show the detail of the Braille pin mechanism. As best seen in the exploded view of FIG. 6A, the top plate 60 of the mouse 10 has a cavity 62 formed therein in which the pins 18 are disposed. The fingers 54 of the pins 18 are confined in guide holes 64 for proper alignment. A leaf spring 66 disposed in a channel 68 engages the upper surfaces of flanges 52 and biases them downward. The upper portions of Braille pins 18 are retained for up-and-down movement in guide holes 70 in the Braille pad 14.
FIGS. 7, 8 and 8A illustrate an alternative embodiment of the actuating mechanism of this invention. In this embodiment, the character indicia 50 (which, as in the first-described embodiment, may be recessed or protruding) are positioned on a flexible belt 72 extending over a drive pulley 74 driven by the stepper motor 26, and an idler pulley 76. The belt 72 is provided with teeth 78 which engage teeth on the drive pulley 74 to prevent the belt 72 from slipping with respect to the drive pulley 74. As in the first-described embodiment, the pressure plate 82 on level arm 34 of the solenoid 36 presses the belt 72 into engagement with the Braille pins 18 when the solenoid 36 is energized.
FIGS. 9, 10 and 10A show a third embodiment of the pin actuating mechanism of this invention, and also illustrate a method, applicable in principle to all three embodiments, of reducing the power consumption and heat generation of solenoid 36. In this third embodiment, the stepper motor 26 is pivotably supported on a swivel pin 84 in a swivel bracket 86. The swivel bracket 86 in this embodiment fixedly supports the solenoid 36. A stiff character drum 90 is fixedly mounted on the shaft 92 of stepper motor 26. The character indicia 50 are carried on the surface of drum 90.
Normally, the spring 88 biases the character drum 90 against the Braille pins 18 contained in the pad 14. When the solenoid 36 is energized, it pushes the actuator 94 to the left in FIG. 9. This causes the inclined surface 96 of actuator 94 to push the wedge arm 98 attached to stepper motor 26 downwardly against the bias of spring 88, thereby pivoting the motor 26 counterclockwise about the swivel pin 84 and pulling the character drum 90 out of engagement with pins 18. Thus, the character drum 90 can be freely rotated to another character position while the solenoid 36 is energized.
The embodiment of FIGS. 9, 10 and 10A has two advantages: the provision of a stiff character drum 90 reduces the risk of character element failure due to flexing fatigue, and the energizing of the solenoid 36 during indexing rather than during character display prevents the continuous energization of the solenoid 36 if the cursor is left on a character of the computer screen. Also, the indexing time from one character to the next is statistically shorter, on the average, than the dwell time on a character being read by the operator.
|Cited Patent
|Filing date
|Publication date
|May 24, 1985
|May 12, 1987
|Oleg Tretiakoff
|Tactile reading device
|Sep 23, 1980
|May 6, 1982
|Claus Colani
|Braille element for alphanumeric output - has pins raised by electro-hydraulic or piezoelectric actuation element forming part of data processing system
|Nov 27, 1980
|Jul 8, 1982
|Licentia Gmbh
|EM controlled pins for braille system - have alternate pole pieces and magnets and slide inside holders containing EM coils
|Feb 26, 1987
|Sep 8, 1988
|Friedrich Horst Ing Papenmeier
|Elektromechanische braille-zeile
|Feb 9, 1988
|Aug 31, 1988
|Marcel Simonin
|Device for displaying relief symbols, in particular Braille characters
|Title not available
|Citing Patent
|Filing date
|Publication date
|Jul 26, 1993
|Nov 14, 1995
|Thompson; James M.
|Braille board with movable dot pins
|Nov 14, 1994
|Aug 19, 1997
|Lucent Technologies Inc.
|Computing interface system
|Nov 14, 1994
|Jan 6, 1998
|Lucent Technologies Inc.
|Computing and telecommunications interface system
|Jan 9, 1997
|Jun 15, 1999
|Virtouch Ltd.
|Mouse-like input/output device with display screen and method for its use
|Oct 21, 1998
|May 9, 2000
|Murphy; Kevin C.
|Tactile recognition input device and overlay for use with an input device
|Aug 3, 1994
|Aug 29, 2000
|Caretec Gmbh
|Device for representing relief items
|Jun 14, 1999
|Aug 21, 2001
|Virtouch, Ltd.
|Tactile interface system for electronic data display system
|Jun 14, 2000
|Jul 13, 2004
|Virtouch Ltd.
|Tactile interface system for electronic data display system
|May 16, 2000
|Aug 17, 2004
|United States Of America
|Refreshable braille reader
|Jan 28, 2003
|Nov 16, 2004
|Dwain Gipson
|Scanning braille presentation
|Jul 31, 2002
|Jan 17, 2006
|Freedom Scientific
|Navigation aid for refreshable braille display and other text products for the vision impaired
|Jan 2, 2003
|Mar 7, 2006
|United States Of America
|Extended refreshable tactile graphic array for scanned tactile display
|Dec 30, 2003
|Mar 28, 2006
|Purdue Research Foundation
|Apparatus and methods for a shape memory spring actuator and display
|Dec 12, 2002
|Apr 1, 2008
|United States Of America
|Refreshable scanning tactile graphic display for localized sensory stimulation
|Jul 27, 2009
|Dec 31, 2013
|Immersion Corporation
|System and method for low power haptic feedback
|Jun 30, 2013
|May 24, 2016
|International Business Machines Corporation
|Voice output device, information input device, file selection device, telephone set, and program and recording medium of the same
|Dec 12, 2002
|Jun 26, 2003
|Roberts John W.
|Refreshable scanning tactile graphic display for localized sensory stimulation
|Jul 31, 2002
|Jul 3, 2003
|Romeo Michael C.
|Navigation aid for refreshable braille display and other text products for the vision impaired
|Jan 2, 2003
|Aug 14, 2003
|Roberts John W.
|Extended refreshable tactile graphic array for scanned tactile display
|Jan 28, 2003
|Jul 29, 2004
|Dwain Gipson
|Scanning braille presentation
|Dec 30, 2003
|Mar 31, 2005
|Schleppenbach David A.
|Apparatus and methods for a shape memory spring actuator and display
|Oct 8, 2004
|Apr 14, 2005
|Askk Co., Ltd.
|Braille display system
|Nov 19, 2009
|Immersion Corporation
|System and method for low power haptic feedback
|May 19, 2011
|Immersion Corporation
|Systems and Methods For A Friction Rotary Device For Haptic Feedback
|Oct 8, 2004
|Apr 13, 2005
|Askk Co.,Ltd.
|Braille display system
|Dec 25, 1997
|Jul 16, 1998
|Virtouch Ltd.
|Mouse-like input/output device with display screen and method for its use
|Jun 14, 2000
|Dec 21, 2000
|Virtouch Ltd.
|Tactile interface system for electronic data display system
|Jun 14, 2000
|Feb 22, 2001
|Virtouch Ltd
|Tactile interface system for electronic data display system
|U.S. Classification
|434/114, 434/112, 434/113
|International Classification
|Cooperative Classification
|European Classification
|May 15, 1991
Owner name: NIMBUS, INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:LE BLANC, PIETER W. C. J.;MAHER, TIMOTHY R.;REEL/FRAME:005718/0590
Effective date: 19910503
|Oct 29, 1996
|Maintenance fee reminder mailed
|Mar 24, 1997
|Fee payment
Year of fee payment: 4
|Mar 24, 1997
|Surcharge for late payment
|Apr 21, 1997
Owner name: NMB ACQUISITION CORPORATION, MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NIMBUS, INC.;REEL/FRAME:008454/0973
Effective date: 19961224
|Feb 3, 1998
Owner name: PIETER W.C.J. LE BLANC, CALIFORNIA
Free format text: RE-ASSIGNMENT;ASSIGNOR:NIMBUS INC;REEL/FRAME:008975/0207
Effective date: 19970513
|Oct 17, 2000
|Maintenance fee reminder mailed
|Mar 25, 2001
|Lapse for failure to pay maintenance fees
|May 29, 2001
|Expired due to failure to pay maintenance fee
Effective date: 20010323
|Oct 2, 2001
Owner name: THORATEC CORPORATION, CALIFORNIA
Free format text: MERGER;ASSIGNOR:THORATEC CARDIOSYSTEMS, INC.;REEL/FRAME:012243/0160
Effective date: 20010629
Owner name: THORATEC CARDIOSYSTEMS INC., CALIFORNIA
Free format text: MERGER;ASSIGNOR:NIMBUS, INC.;REEL/FRAME:012243/0178
Effective date: 20010618


text:
iHerb カスタマーレビュー
のセーブ: ¥24 (5 % オフ)
Great price!
Reviewer1469615により Feb 20, 2009 に掲載
Tasty product-I now sprinkle it on everything (yogurt,hot cereals,meat loaf,smoothies,etc). iHerb's price=$2.50 less than Whole Foods--and this package is light(keeping postage costs down). I'm very pleased.
Great and easy way to add fiber to your life!
Reviewer2001475により May 14, 2009 に掲載
I simple sprinkle two TBS on top of my oatmeal every morning and feel good about increasing my fiber intake. After opening, I pour much of it into a smaller plastic container for easier handling and refrigerate both. Lots of other ways to use this too if you're into baking.
For Oatmeal
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I mix this in my steelcut oats for extra fiber and Omega3.
Bob's Red Mill Flaxseed Meal
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I am never disappointed by Bob. I use this in recipes and add it to oatmeal and things for extra fiber, protein, and omega-3s.
Yum and regular.
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I sprinkle this on my cereal every day to stay regular. It is also supposed to lower your cholesterol. Good value and quality.
the best
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i love all all bob's red mill product.
Makes Great Flatbread, Muffins and Pancakes
Reviewer1261830により Oct 07, 2010 に掲載
Besides adding flax meal to my oatmeal and smoothies, I enjoy making delicious flatbread, pancakes and muffins from it. My flatbreads can be a little sweet if I desire with a touch of honey added, or seasoned with herbs and spices for great sandwiches. Flax meal is a great way to get my fiber and Omega-3s and this is a good brand. Thanks iHerb!
4613142442863848488により Jul 21, 2012 に掲載
I used this in cooking and had a few teaspoonfuls raw, very good for you. Also gave some to my Golden Retriever, he loved it and his coat is looking much nicer.
Great product.
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I add this to my bread dough when baking. It adds some crunch.
Bob's Red Mill, Whole Ground Flaxseed Meal
Reviewer2021726により Apr 25, 2010 に掲載
Shipping time was very good. While this product appears to make for a better alternative than the Metamucil I was using before it.