New! View global litigation for patent families

US20040121314A1 - Methods for rapid detection and identification of bioagents in containers - Google Patents

Methods for rapid detection and identification of bioagents in containers Download PDF

Info

Publication number
US20040121314A1
US20040121314A1 US10326642 US32664202A US2004121314A1 US 20040121314 A1 US20040121314 A1 US 20040121314A1 US 10326642 US10326642 US 10326642 US 32664202 A US32664202 A US 32664202A US 2004121314 A1 US2004121314 A1 US 2004121314A1
Authority
US
Grant status
Application
Patent type
Prior art keywords
mass
base
nucleic
acid
method
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10326642
Inventor
David Ecker
Richard Griffey
Rangarajan Sampath
Steven Hofstadler
John McNeil
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ibis Biosciences Inc
Original Assignee
Ionis Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions

Abstract

Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a “base composition signature” (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.

Description

    STATEMENT OF GOVERNMENT SUPPORT
  • [0001] This invention was made with United States Government support under DARPA/SPO contract BAA00-09. The United States Government may have certain rights in the invention.
  • FIELD OF THE INVENTION
  • [0002]
    The present invention relates to methods for rapid detection and identification of bioagents from environmental, clinical or other samples. The methods provide for detection and characterization of a unique mass or unique base composition signature (BCS) from any bioagent, including bacteria and viruses. The unique mass or unique BCS is used to rapidly identify the bioagent(s).
  • BACKGROUND OF THE INVENTION
  • [0003]
    Rapid and definitive microbial identification is desirable for a variety of industrial, medical, environmental, quality, and research reasons. Traditionally, the microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through direct examination and culture of specimens. Since the mid-1980s, researchers have repeatedly demonstrated the practical utility of molecular biology techniques, many of which form the basis of clinical diagnostic assays. Some of these techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification Of nucleic acids (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). These procedures, in general, are time-consuming and tedious. Another option is the polymerase chain reaction (PCR) or other amplification procedure that amplifies a specific target DNA sequence based on the flanking primers used. Finally, detection and data analysis convert the hybridization event into an analytical result.
  • [0004]
    Other techniques for detection of bioagents include high-resolution mass spectrometry (MS), low-resolution MS, fluorescence, radioiodination, DNA chips and antibody techniques. None of these techniques is entirely satisfactory.
  • [0005]
    Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. However, high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents (“cluttered” background). Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
  • [0006]
    Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
  • [0007]
    Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem. 69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998; Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
  • [0008]
    U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences that comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
  • [0009]
    U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
  • [0010]
    WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
  • [0011]
    WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand, which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
  • [0012]
    PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
  • [0013]
    U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
  • [0014]
    WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
  • [0015]
    WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
  • [0016]
    Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need.
  • SUMMARY OF THE INVENTION
  • [0017]
    One embodiment of the present invention is a method of identifying an unknown bioagent in a container comprising a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers that hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; b) amplifying the variable nucleic acid sequence to produce an amplification product; c) determining the molecular mass of the amplification product; and d) comparing the molecular mass to one or more molecular masses of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein a match identifies the unknown bioagent in the container. In some embodiments, the container is a box or envelope. In one aspect of the invention, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of the invention, the bioagent is a bacterium, virus, parasite, fungi, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps a)-d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps a)-c) on a different plurality of known organisms from those in step d). Preferably, the one or more molecular mass is contained in a database of molecular masses. In another aspect of the invention, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
  • [0018]
    Another embodiment of the present invention is a method of identifying an unknown bioagent in a container comprising a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers that hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; b) amplifying the variable nucleic acid sequence to produce an amplification product; c) determining the base composition of the amplification product; and d) comparing the base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein a match identifies the unknown bioagent in the container. In some embodiments, the container is a box or envelope. In one aspect of the invention, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of the invention, the bioagent is a bacterium, virus, parasite, fungi, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps a)-d) using a different oligonucleotide primer pair and comparing the results to one or more base composition signatures of amplification products obtained by performing steps a)-c) on a different plurality of known organisms from those in step d). Preferably, the one or more base compositions is contained in a database of base compositions. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • [0019]
    FIGS. 1A-1H and FIG. 2 are consensus diagrams that show examples of conserved regions from 16S rRNA (FIG. 1A, 1A-2, 1A-3, 1A-4, 1B, 1B-1, and 1B-2), 23S rRNA (3′-half, FIGS. 1C-1, 1C-2, and 1D; 5′-half, FIGS. 1E-F), 23S rRNA Domain I (FIG. 1G), 23S rRNA Domain IV (FIG. 1H) and 16S rRNA Domain III (FIG. 2) which are suitable for use in the present invention. Where there is overlap or redundancy between the figures, the overlap is simply provided as an orientation aid and no additional members of the sequence are implied thereby. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. Bases in capital letters are greater than 95% conserved; bases in lower case letters are 90-95% conserved, filled circles are 80-90% conserved; and open circles are less than 80% conserved. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. The nucleotide sequence of the 16S rRNA consensus sequence is SEQ ID NO:3 and the nucleotide sequence of the 23S rRNA consensus sequence is SEQ ID NO:4.
  • [0020]
    [0020]FIG. 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in FIG. 1C.
  • [0021]
    [0021]FIG. 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved.
  • [0022]
    [0022]FIG. 4 is a schematic diagram of base composition signature determination using nucleotide analog “tags” to determine base composition signatures.
  • [0023]
    [0023]FIG. 5 shows the deconvoluted mass spectra of a Bacillus anthracis region with and without the mass tag phosphorothioate A (A*). The two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
  • [0024]
    [0024]FIG. 6 shows base composition signature (BCS) spectra from PCR products from Staphylococcus aureus (S. aureus 16S1337F) and Bacillus anthracus (B. anthr. 16S1337F), amplified using the same primers. The two strands differ by only two (ATE→CG) substitutions and are clearly distinguished on the basis of their BCS.
  • [0025]
    [0025]FIG. 7 shows that a single difference between two sequences (A14 in B. anthracis vs. A15 in B. cereus) can be easily detected using ESI-TOF mass spectrometry.
  • [0026]
    [0026]FIG. 8 is an ESI-TOF of Bacillus anthracis spore coat protein sspE 56mer plus calibrant. The signals unambiguously identify B. anthracis versus other Bacillus species.
  • [0027]
    [0027]FIG. 9 is an ESI-TOF of a B. anthracis synthetic 16S1228 duplex (reverse and forward strands). The technique easily distinguishes between the forward and reverse, strands.
  • [0028]
    [0028]FIG. 10 is an ESI-FTICR-MS of a synthetic B. anthracis 16S1337 46 base pair duplex.
  • [0029]
    [0029]FIG. 11 is an ESI-TOF-MS of a 56mer oligonucleotide (3 scans) from the B. anthracis saspB gene with an internal mass standard. The internal mass standards are designated by asterisks.
  • [0030]
    [0030]FIG. 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFA buffer showing that charge stripping with tributylammonium trifluoroacetate reduces the most abundant charge state from [M-8H+]8− to [M-3H+]3−.
  • DETAILED DESCRIPTION OF EMBODIMENTS
  • [0031]
    The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with PCR-based BCS or mass technology using “intelligent primers” which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The high-resolution MS technique is used to determine the molecular mass and/or base composition signature (BCS) of the amplified sequence region. This unique mass or unique “base composition signature” (BCS) is then input to a maximum-likelihood detection algorithm for matching against a database of masses or base composition signatures in the same amplified region. The present method combines PCR-based amplification technology (which provides specificity) and a molecular mass detection mode (which provides speed and does not require nucleic acid sequencing of the amplified target sequence) for bioagent detection and identification.
  • [0032]
    The present method allows extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure. The method leverages ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives. Thus, the methods are useful in a wide variety of fields, including, but not limited to, those fields discussed below.
  • [0033]
    In some embodiments of the invention, the methods disclosed herein can be used for environmental testing. Detection and discrimination of pathogenic vs. non-pathogenic bacteria, viruses, parasites, fungi and the like, in samples of water, land, air, or other samples, can be carried out. Water samples can be obtained from, for example, lakes, rivers, oceans, streams, water treatment systems, rainwater, groundwater, water table, reservoirs, wells, bottled water, and the like. Air samples can be obtained from ventilation systems, airplane cabins, schools, hospitals, mass transit locations such as subways, train stations, airports, and the like. Conditions such as sick building syndrome can be detected. Land samples can be obtained from any location.
  • [0034]
    In other embodiments of the invention, the methods disclosed herein can be used to screen blood and other bodily fluids and tissues for pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like. Animal samples, including but not limited to, blood and other bodily fluid and tissue samples, can be obtained from living animals, who are either known or not known to or suspected of having a disease, infection, or condition. Alternately, animal samples such as blood and other bodily fluid and tissue samples can be obtained from deceased animals. Blood samples can be further separated into plasma or cellular fractions and further screened as desired. Bodily fluids and tissues can be obtained from any part of the animal or human body. Animal samples can be obtained from, for example, mammals and humans.
  • [0035]
    In other embodiments of the invention, the methods disclosed herein can be used for forensics. For example, medical examiners can use the present invention to determine the cause of death. In addition, epidemiologists, for example, can use the present methods to determine the geographic origin of a particular strain of bacteria or virus. For example, a particular strain of bacteria or virus may have a sequence difference that is associated with a particular area of a country or the world and identification of such a sequence difference can lead to the identification of the geographic origin and epidemiological tracking of the spread of the particular disease, disorder or condition associated with the detected virus or bacteria. In addition, carriers of particular DNA or diseases, such as mammals, non-mammals, birds, insects, and plants, can be tracked by screening SNPs, VNTRs, or polyA, for example. Diseases, such as malaria, can be tracked by screening commensals, such as mosquitos.
  • [0036]
    In other embodiments of the invention, the methods disclosed herein can be used for detecting the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like in samples of foodstuff or cosmetics. For example, food and wine can be examined for the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like. Particular types of foods susceptible to bioagent contamination, such as agricultural products, meat products and eggs, can be examined for pathogenic organisms such as E. coli and Salmonella species. Such examination procedures can be used by, for example, the wholesalers of foodstuffs and beverages, or by regulatory agencies such as the U.S. Department of Agriculture and the Food and Drug Administration. In addition, grapes and wines, for example, can be examined using the present methods to detect particular strains of bacteria or yeast that may indicate a particular time upon which to harvest the grapes or alter the wine-making process.
  • [0037]
    In other embodiments of the invention, the methods disclosed herein can be used for detecting the presence of bioagents in a container, such as a package, box, envelope, mail tube, railroad box car, and the like. For example, mail and package delivery entities and agencies, both domestic and abroad, as well investigative agencies such as the FBI and ATF can use the present methods to detect bioagents in containers.
  • [0038]
    In other embodiments of the invention, the methods disclosed herein can be used for detecting the presence of pathogenic and non-pathogenic bacteria, viruses, parasites, fungi and the like in organ donors and/or in organs from donors. Such examination can result in the prevention of the transfer of, for example, viruses such as West Nile virus, hepatitis viruses, human immunodeficiency virus, and the like from a donor to a recipient via a transplanted organ. The methods disclosed herein can also be used for detection of host versus graft or graft versus host rejection issues related to organ donors by detecting the presence of particular antigens in either the graft or host known or suspected of causing such rejection. In particular, the bioagents in this regard are the antigens of the major histocompatibility complex, such as the HLA antigens.
  • [0039]
    In other embodiments of the invention, the methods disclosed herein can be used for detection and identification of livestock infections such as, for example, mad cow disease, hoof and mouth disease, and the like. Livestock includes, but is not limited to, cows, pigs, sheep, chickens, turkeys, goats, and other farm animals.
  • [0040]
    In other embodiments of the invention, the methods disclosed herein can be used for pharmacogenetic analysis and medical diagnosis including, but not limited to, cancer diagnosis based on mutations and polymorphisms, drug resistance and susceptibility testing, screening for and/or diagnosis of genetic diseases and conditions, and diagnosis of infectious diseases and conditions. The present methods can also be used to detect and track emerging infectious diseases, such as West Nile virus infection, mad cow disease, and HIV-related diseases.
  • [0041]
    The present methods can be used to detect and classify any biological agent, including bacteria, viruses, fungi and toxins. As one example, where the agent is a biological threat, the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes. In addition, the methods can be used to identify natural or deliberate engineering events including chromosome Fragment swapping, molecular breeding (gene shuffling) and emerging infectious diseases.
  • [0042]
    Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species (Proc. Nall. Acad. Sci. U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins are DNA polymerase III beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G. Operons can also be targeted using the present method. One example of an operon is the bfp operon from enteropathogenic E. coli. Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential. The method can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
  • [0043]
    A theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor. The complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved. However, the present invention provides a straightforward strategy for obtaining information with the same practical value using base composition signatures (BCS). While the base composition of a gene fragment is not as information-rich as the sequence itself, there is no need to analyze the complete sequence of the gene if the short analyte sequence fragment is properly chosen. A database of reference sequences can be prepared in which each sequence is indexed to a unique base composition signature, so that the presence of the sequence can be inferred with accuracy from the presence of the signature. The advantage of base composition signatures is that they can be quantitatively measured in a massively parallel fashion using multiplex PCR (PCR in which two or more primer-pairs amplify target sequences simultaneously) and mass spectrometry. These multiple primer amplified regions uniquely identify most threat and ubiquitous background bacteria and viruses. In addition, cluster-specific primer pairs distinguish important local clusters (e.g., anthracis group).
  • [0044]
    In the context of this invention, a “bioagent” is any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, parasites, fungi, toxin genes and bioregulating compounds. Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.
  • [0045]
    As used herein, a “base composition signature” (BCS) is the exact base composition from selected fragments of nucleic acid sequences that uniquely identifies the target gene and source organism. BCS can be thought of as unique indexes of specific genes.
  • [0046]
    As used herein, “intelligent primers” are primers which bind to sequence regions which flank an intervening variable region. In a preferred embodiment, these sequence regions which flank the variable region are highly conserved among different species of bioagent. For example, the sequence regions may be highly conserved among all Bacillus species. By the term “highly conserved”, it is meant that the sequence regions exhibit between about 80-100%, more preferably between about 90-100% and most preferably between about 95-100% identity. Examples of intelligent primers which amplify regions of the 16S and 23S rRNA are shown in FIGS. 1A-1I. A typical primer amplified region in 16S rRNA is shown in FIG. 2. The arrows represent primers which bind to highly conserved regions which flank a variable region in 16S rRNA domain III. The amplified region is the stem-loop structure under “1100-11 88.”
  • [0047]
    One main advantage of the detection methods of the present invention is that the primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same primer pair can be used to identify any desired bacterium because it will bind to the conserved regions which flank a variable region specific to a single species, or common to several bacterial species, allowing nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition. For example, the 16S971-1062, 16S1228-1310 and 16S1100-1188 regions are 98-99% conserved in about 900 species of bacteria (16S=16S rRNA, numbers indicate nucleotide position). In one embodiment of the present invention, primers used in the present method bind to one or more of these regions or portions thereof.
  • [0048]
    The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with nucleic acid amplification-based BCS technology using “intelligent primers” which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s). Although the use of PCR is preferred, other nucleic acid amplification techniques may also be used, including ligase chain reaction (LCR) and strand displacement amplification (SDA). The high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments. The resolved spectral lines are then translated to BCS which are input to a maximum-likelilhood detection algorithm matched against spectra for one or more known BCS. Preferably, the bioagent BCS spectrum is matched against one or more databases of BCS from vast numbers of bioagents. Preferably, the matching is done using a maximum-likelihood detection algorithm.
  • [0049]
    In a preferred embodiment, base composition signatures are quantitatively measured in a massively parallel fashion using the polymerase chain reaction (PCR), preferably multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids must be present for detection of bioagents by MS. A wide variety of techniques for preparing large amounts of purified nucleic acids or fragments thereof are well known to those of skill in the art. PCR requires one or more pairs of oligonucleotide primers which bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer. The primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Taq polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis. The solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence.
  • [0050]
    The “intelligent primers” define the target sequence region to be amplified and analyzed. In one embodiment, the target sequence is a ribosomal RNA (rRNA) gene sequence. With the complete sequences of many of the smallest microbial genomes now available, it is possible to identify a set of genes that defines “minimal life” and identify composition signatures that uniquely identify each gene and organism. Genes that encode core life functions such as DNA replication, transcription, ribosome structure, translation, and transport are distributed broadly in the bacterial genome and are preferred regions for BCS analysis. Ribosomal RNA (rRNA) genes comprise regions that provide useful base composition signatures. Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes. For example, there are currently over 13,000 sequences in the ribosomal RNA database that has been created and maintained by Robin Gutell, University of Texas at Austin, and is publicly available on the Institute for Cellular and Molecular Biology web page (www.rna.icmb.utexas.edu/). There is also a publicly available rRNA database created and maintained by the University of Antwerp, Belgium at www.rrna.uia.ac.be.
  • [0051]
    These databases have been analyzed to determine regions that are useful as base composition signatures. The characteristics of such regions are: a) between about 80 and 100%, preferably >about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, preferably no more than about 50-250 nucleotides, and more preferably no more than about 60-100 nucleotides, between the conserved regions.
  • [0052]
    Due to their overall conservation, the flanking rRNA primer sequences serve as good “universal” primer binding sites to amplify the region of interest for most, if not all, bacterial species. The intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature.
  • [0053]
    It is advantageous to design the “intelligent primers” to be as universal as possible to minimize the number of primers which need to be synthesized, and to allow detection of multiple species using a single pair of primers. These primer pairs can be used to amplify variable regions in these species. Because any variation (due to codon wobble in the 3rd position) in these conserved regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a “universal base”. For example, under this “wobble” pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res. 24:3302-3306, 1996).
  • [0054]
    In another embodiment of the invention, to compensate for the somewhat weaker binding by the “wobble” base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs are 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G. Propynes are described in U.S. Pat. Nos. 5,645,985, 5,830.653 and 5,484,908, the entire contents of which are incorporated herein by reference. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, the entire contents of which are incorporated herein by reference. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, the entire contents of which are incorporated herein by reference.
  • [0055]
    In some embodiments of the invention, the methods described herein can be used for defense of germ warfare (allowing immediate identification of the bioagent and appropriate treatment). Bacterial biological warfare agents capable of being detected by the present methods include Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders), Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, and Vibrio cholerae.
  • [0056]
    Besides 16S and 23S rRNA, other target regions suitable for use in the present invention for detection of bacteria include 55 rRNA and RNase P (FIG. 3).
  • [0057]
    Biological warfare fungus biowarfare agents include coccidioides immitis (Coccidioidomycosis).
  • [0058]
    Biological warfare toxin genes capable of being detected by the methods of the present invention include botulism, T-2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin and saxitoxin.
  • [0059]
    Biological warfare viral threat agents are mostly RNA viruses (positive-strand and negative-strand), with the exception of smallpox. Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high. RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases.
  • [0060]
    Examples of (−)-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbillivirus).
  • [0061]
    Examples of (+)-strand RNA viruses include picornaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross River virus, Venezuelan equine encephalitis virus, Rubella virus, hepatitis E virus). The hepatitis C virus has a 5′-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3′-untranslated region of 240 nucleotides. The 5′-UTR and 3′-UTR are 99% conserved in hepatitis C viruses.
  • [0062]
    In one embodiment, the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a (−)-strand RNA virus. (+)-strand RNA viruses are double stranded RNA and replicate by RNA-directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template. Helicase unwinds the RNA duplex to allow replication of the single stranded RNA. These viruses include viruses from the family picornaviridae (e.g., poliovirus, coxsackievirus, echovirus), togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae (e.g., lymphocytic choriomeningitis virus, lassa fever virus), cononaviridae (e.g., human respiratory virus) and Hepatitis A virus. The genes encoding these proteins comprise variable and highly conserved regions which flank the variable regions.
  • [0063]
    In a preferred embodiment, the detection scheme for the PCR products generated from the bioagent(s) incorporates three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a BCS that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel.
  • [0064]
    In one embodiment, the method can be used to detect the presence of antibiotic resistance and/or toxin genes in a bacterial species. For example, Bacillus anthracis comprising a tetracycline resistance plasmid and plasmids encoding one or both anthracis toxins (px01 and/or px02) can be detected by using antibiotic resistance primer sets and toxin gene primer sets. If the B. anthracis is positive for tetracycline resistance, then a different antibiotic, for example quinalone, is used.
  • [0065]
    Mass spectrometry (MS)-based detection of PCR products provides all of these features with additional advantages. MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product with a unique base composition is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons. Intact molecular ions can be generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). For example, MALDI of nucleic acids, along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.).
  • [0066]
    In some embodiments, large DNAs and RNAs, or large amplification products therefrom, can be digested with restriction endonucleases prior to ionization. Thus, for example, an amplification product that was 10 kDa could be digested with a series of restriction endonucleases to produce a panel of, for example, 100 Da fragments. Restriction endonucleases and their sites of action are well known to the skilled artisan. In this manner, mass spectrometry can be performed for the purposes of restriction mapping.
  • [0067]
    Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
  • [0068]
    The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.
  • [0069]
    In general, the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS). In one embodiment of the invention, the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents). To achieve minimal latency, a continuous sample stream flows directly into the PGC-MS combustion chamber. When an increase in biomass is detected, a PCR process is automatically initiated. Bioagent presence produces elevated levels of large molecular fragments from 100-7,000 Da which are observed in the PGC-MS spectrum. The observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove(combining PCR and MS, preferably FT-ICR MS) is initiated. Optionally, alarms or other processes (halting ventilation flow, physical isolation) are also initiated by this detected biomass level.
  • [0070]
    The accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance 13C and 15N isotopes, and assignment of the charge state for any ion. The cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow. The latter two problems are addressed by operating with a resolving power of >100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA. The resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [M-14H+]14− charge state of an 84mer PCR product is poorly characterized and assignment of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components arc visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward. The [13C15N]-depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al., Nucl. Acids Res. 20:4515-4523, 1992).
  • [0071]
    While mass measurements of intact nucleic acid regions are believed to be adequate to determine most bioagents, tandem mass spectrometry (MSn) techniques may provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For-example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3′ terminus and a 5′ phosphate following internal cleavage) and the a-Base series(having an intact 5′ terminus and a 3′ furan).
  • [0072]
    The second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample.
  • [0073]
    If there are two or more targets of similar base composition or mass, or if a single amplification reaction results in a product which has the same mass as two or more bioagent reference standards, they can be distinguished by using mass-modifying “tags.” In this embodiment of the invention, a nucleotide analog or “tag” is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses. Such tags are described in, for example, PCT WO97/33000. This further limits the number of possible base compositions consistent with any mass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined.
  • [0074]
    In another amplification reaction, the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C. The combination of the A/T reaction and G/C reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in FIG. 4 and Table 1.
    TABLE 1
    Total
    Total Base Base base Total base
    Δmass info info comp. comp.
    Double strand Single strand this this other Top Bottom
    Mass tag sequence Sequence strand strand strand strand strand
    T*.Δmass T*ACGT*ACGT* T*ACGT*ACGT 3x 3T 3A 3T 3A
    (T* − T) = x AT*GCAT*GCA 2A 2T
    2C 2G
    2G 2C
    AT*GCAT*GCA 2x 2T 2A
    C*.Δmass TAC*GTAC*GT TAC*GTAC*GT 2x 2C 2G
    (C* − C) = y ATGC*ATGC*A
    ATGC*ATGC*A 2x 2C 2G
  • [0075]
    The mass tag phosphorothioate A (A*) was used to distinguish a Bacillus anthracis cluster. The B. anthracis (A14G9C14T9) had an average MW of 14072.26, and the B. anthracis (A1A*13G9C14T9) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS. The deconvoluted spectra are shown in FIG. 5.
  • [0076]
    In another example, assume the measured molecular masses of each strand are 30,000.115 Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand.
  • [0077]
    Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing. The detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes.
  • [0078]
    Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels. The program emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database (e.g. GenBank) is used to define the mass basecount matched filters. The database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. the maximum likelihood process is applied to this “cleaned up” data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.
  • [0079]
    In one embodiment, a strategy to “triangulate” each organism by measuring signals from multiple core genes is used to reduce false negative and false positive signals, and enable reconstruction of the origin or hybrid or otherwise engineered bioagents. After identification of multiple core genes, alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and potential primer binding sites flanking variable regions are identified. Next, amplification target regions for signature analysis are selected which distinguishes organisms based on specific genomic differences (i.e., base composition). For example, detection of signatures for the three part toxin genes typical of B. anthracis (Bowen, J. E. and C. P. Quinn, J. Appl. Microbiol. 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
  • [0080]
    The present method can also be used to detect single nucleotide polymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly and accurately. A SNP is defined as a single base pair site in the genome that is different from one individual to another. The difference can be expressed either as a deletion, an insertion or a substitution, and is frequently linked to a disease state. Because they occur every 100-1000 base pairs, SNPs are the most frequently bound type of genetic marker in the human genome.
  • [0081]
    For example, sickle cell anemia results from an A-T transition, which encodes a valine rather than a glutamic acid residue. Oligonucleotide primers may be designed such that they bind to sequences that flank a SNP site, followed by nucleotide amplification and mass determination of the amplified product. Because the molecular masses of the resulting product from an individual who does not have sickle cell anemia is different from that of the product from an individual who has the disease, the method can be used to distinguish the two individuals. Thus, the method can be used to detect any known SNP in an individual and thus diagnose or determine increased susceptibility to a disease or condition.
  • [0082]
    In one embodiment, blood is drawn from an individual and peripheral blood mononuclear cells (PBMC) are isolated and simultaneously tested, preferably in a high-throughput screening method, for one or more SNPs using appropriate primers based on the known sequences which flank the SNP region. The National Center for Biotechnology Information maintains a publicly available database of SNPs (www.ncbi.nlm.nih.gov/SNP/).
  • [0083]
    The method of the present invention can also be used for blood typing. The gene encoding A, B or O blood type can differ by four single nucleotide polymorphisms. If the gene contains the sequence CGTGGTGACCCTT (SEQ ID NO:5), antigen A results. If the gene contains the sequence CGTCGTCACCGCTA (SEQ ID NO:6) antigen B results. If the gene contains the sequence CGTGGT-ACCCCTT (SEQ ID NO:7), blood group O results (“—” indicates a deletion). These sequences can be distinguished by designing a single primer pair that flanks these regions, followed by amplification and mass determination.
  • [0084]
    In other embodiments of the invention, all of the aformentioned methods can be used to determine or confirm the absence of a bioagent in a sample. For example, when the molecular mass or base composition is compared to one or more molecular masses or base compositions obtained from a plurality of known organisms and no match is obtained, the absence of a particular bioagent is determined or confirmed. In these methods of determining the absence of a bioagent, a positive control can be used to confirm the integrity of the system. Such positive controls include, but are not limited to addition of a known bioagent to the sample and confirming the presence of the known bioagent in the sample by obtaining a match from the comparison step. Alternately, residual primer signal can be detected, thus indicating that the system integrity is intact.
  • [0085]
    While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.
  • EXAMPLES Example 1 Nucleic Acid Isolation and PCR
  • [0086]
    In one embodiment, nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry. Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al.). The nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
  • Example 2 Mass Spectrometry
  • [0087]
    FTICR Instrumentation: The FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber. The spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute. The Bruker data-acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments. For oligonucleotides in the 20-30-mer regime typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z.
  • [0088]
    Modified ESI Source: In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator. The ESI ion optics consists of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz. A lab-built electro-mechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the “open” position via a TTL pulse from the data station. When in the “closed” position, a stable electrospray plume is maintained between the ESI emitter and the face of the shutter. The back face of the shutter arm contains an elastomeric seal that can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position. When the shutter is triggered, a “time slice” of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir. The rapid response time of the ion shutter (<25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir.
  • [0089]
    Apparatus for Infrared Multiphoton Dissociation A 25 watt CW CO2 laser operating at 10.6/m has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications. An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet. Using standard IR-compatible mirrors and kinematic mirror mounts, the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
  • Example 3 Identification of Bioagents
  • [0090]
    Table 1 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms. The primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in FIGS. 1A-1C. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The primer pairs are >95% conserved in the bacterial sequence database (currently over 10,000 organisms). The intervening regions are variable in length and/or composition, thus providing the base composition “signature” (BCS) for each organism. Primer pairs were chosen so the total length of the amplified region is less than about 80-90 nucleotides. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • [0091]
    Included in the short bacterial database cross-section in Table 1 are many well known pathogens/biowarfare agents (shown in bold/red typeface) such as Bacillus anthracis or Yersinia pestis as well as some of the bacterial organisms found commonly in the natural environment such as Streptomyces. Even closely related organisms can be distinguished from each other by the appropriate choice of primers. For instance, two low G+C organisms, Bacillus anthracis and Staph aureus, can be distinguished from each other by using the primer pair defined by 16S1337 or 23S855 (M of 4 Da).
    TABLE 2
    Cross Section Of A Database Of Calculated Molecular Masses1
    Primer Regions
    Bug Name 16S_971 16S_1100 16S_1337 16S_1294 16S_1228 23S_1021 23S_855 23S_193 23S_115
    Acinetobacter calcoaceticus 55619.1 55004 28446.7 35854.9 51295.4 30299 42654 39557.5 54999
    Figure US20040121314A1-20040624-P00805
    55005 54388 28448 35238 51296 30295 42651 39560 56850
    Bacillus cereus 55622.1 54387.9 28447.6 35854.9 51296.4 30295 42651 39560.5 56850.3
    Bordetella bronchiseptica 56857.3 51300.4 28446.7 35857.9 51307.4 30299 42653 39559.5 51920.5
    Borrelia burgdorferi 56231.2 55621.1 28440.7 35852.9 51295.4 30297 42029.9 38941.4 52524.6
    Figure US20040121314A1-20040624-P00820
    58098 55011 28448 35854 50683
    Campylobacter jejuni 58088.5 54386.9 29061.8 35856.9 50674.3 30294 42032.9 39558.5 45732.5
    Figure US20040121314A1-20040624-P00821
    55000 55007 29063 35855 50676 30295 42036 38941 56230
    Figure US20040121314A1-20040624-P00822
    55006 53767 28445 35855 51291 30300 42656 39562 54999
    Clostridium difficile 56855.3 54386.9 28444.7 35853.9 51296.4 30294 41417.8 39556.5 55612.2
    Enterococcus faecalis 55620.1 54387.9 28447.6 35858.9 51296.4 30297 42652 39559.5 56849.3
    Figure US20040121314A1-20040624-P00808
    55622 55009 28445 35857 51301 30301 42656 39562 54999
    Figure US20040121314A1-20040624-P00823
    53769 54385 28445 35856 51298
    Haemophilus influenzae 55620.1 55006 28444.7 35855.9 51298.4 30298 42656 39560.5 55613.1
    Kiebsiella pneumoniae 55622.1 55008 28442.7 35856.9 51297.4 30300 42655 39562.5 55000
    Figure US20040121314A1-20040624-P00824
    55618 55626 28446 35857 51303
    Mycobacterium avium 54390.9 55631.1 29064.8 35858.9 51915.5 30298 42656 38942.4 56241.2
    Mycobacterium leprae 54389.9 55629.1 29064.8 35860.9 51917.5 30298 42656 39559.5 56240.2
    Mycobacterium tuberculosis 54390.9 55629.1 29064.8 35860.9 51301.4 30299 42656 39560.5 56243.2
    Mycoplasma genitalium 53143.7 45115.4 29061.8 35854.9 50671.3 30294 43264.1 39558.5 56842.4
    Mycoplasma pneumoniae 53143.7 45118.4 29061.8 35854.9 50673.3 30294 43264.1 39559.5 56843.4
    Neisseria gonorrhoeae 55627.1 54389.9 28445.7 35855.9 51302.4 30300 42649 39561.5 55000
    Figure US20040121314A1-20040624-P00825
    55623 55010 28443 35858 51301 30298 43272 39558 55619
    Figure US20040121314A1-20040624-P00826
    58093 55621 28448 35853 50677 30293 42650 39559 53139
    Figure US20040121314A1-20040624-P00827
    58094 55623 28448 35853 50679 30293 42648 39559 53755
    Figure US20040121314A1-20040624-P00828
    55622 55005 28445 35857 51301 30301 42658
    Figure US20040121314A1-20040624-P00809
    55623 55009 28444 35857 51301
    Staphylococcus aureus 56854.3 54386.9 28443.7 35852.9 51294.4 30298 42655 39559.5 57466.4
    Streptomyces 54389.9 59341.6 29063.8 35858.9 51300.4 39563.5 56864.3
    Treponema pallidum 56245.2 55631.1 28445.7 35851.9 51297.4 30299 42034.9 38939.4 57473.4
    Figure US20040121314A1-20040624-P00829
    55625 55626 28443 35857 52536 29063 30303 35241 50675
    Vibrio parahaemolyticus 54384.9 55626.1 28444.7 34620.7 50064.2
    Figure US20040121314A1-20040624-P00818
    55620 55626 28443 35857 51299
  • [0092]
    [0092]FIG. 6 shows the use of ESI-FT-ICR MS for measurement of exact mass. The spectra from 46mer PCR products originating at position 1337 of the 16S rRNA from S. aureus (upper) and B. anthracis (lower) are shown. These data are from the region of the spectrum containing signals from the [M-8H+]8− charge states of the respective 5′-3′ strands. The two strands differ by two (AT→CG) substitutions, and have measured masses of 14206.396 and 14208.373±0.010 Da, respectively. The possible base compositions derived from the masses of the forward and reverse strands for the B. anthracis products are listed in Table 3.
    TABLE 3
    Possible base composition for B. anthracis products
    Calc. Mass Error Base Comp.
    14208.2935 0.079520 A1 G17 C10 T18
    14208.3160 0.056980 A1 G20 C15 T10
    14208.3386 0.034440 A1 G23 C20 T2
    14208.3074 0.065560 A6 G11 C3 T26
    14208.3300 0.043020 A6 G14 C8 T18
    14208.3525 0.020480 A6 G17 C13 T10
    14208.3751 0.002060 A6 G20 C18 T2
    14208.3439 0.029060 A11 G8 C1 T26
    14208.3665 0.006520 A11 G11 C6 T18
    14208.3890 0.016020 A11 G14 C11 T10
    14208.4116 0.038560 A11 G17 C16 T2
    14208.4030 0.029980 A16 G8 C4 T18
    14208.4255 0.052520 A16 G11 C9 T10
    14208.4481 0.075060 A16 G14 C14 T2
    14208.4395 0.066480 A21 G5 C2 T18
    14208.4620 0.089020 A21 G8 C7 T10
    14079.2624 0.080600 A0 G14 C13 T19
    14079.2849 0.058060 A0 G17 C18 T11
    14079.3075 0.035520 A0 G20 C23 T3
    14079.2538 0.089180 A5 G5 C1 T35
    14079.2764 0.066640 A5 G8 C6 T27
    14079.2989 0.044100 A5 G11 C11 T19
    14079.3214 0.021560 A5 G14 C16 T11
    14079.3440 0.000980 A5 G17 C21 T3
    14079.3129 0.030140 A10 G5 C4 T27
    14079.3354 0.007600 A10 G8 C9 T19
    14079.3579 0.014940 A10 G11 C14 T11
    14079.3805 0.037480 A10 G14 C19 T3
    14079.3494 0.006360 A15 G2 C2 T27
    14079.3719 0.028900 A15 G5 C7 T19
    14079.3944 0.051440 A15 G8 C12 T11
    14079.4170 0.073980 A15 G11 C17 T3
    14079.4084 0.065400 A20 G2 C5 T19
    14079.4309 0.087940 A20 G5 C10 T13
  • [0093]
    Among the 16 compositions for the forward strand and the 18 compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary, corresponding to the actual base compositions of the B. anthracis PCR products.
  • Example 4 BCS of Region from Bacillus anthracis and Bacillus cereus
  • [0094]
    A conserved Bacillus region from B. anthracis (A14G9C14T9) and B. cereus (A15G9C13T9) having a C to A base change was synthesized and subjected to ESI-TOF MS. The results are shown in FIG. 7 in which the two regions are clearly distinguished using the method of the present invention (MW=14072.26 vs. 14096.29).
  • Example 5 Identification of Additional Bioagents
  • [0095]
    In other examples of the present invention, the pathogen Vibrio cholera can be distinguished from Vibrio parahemolyticus with ΔM>600 Da using one of three 16S primer sets shown in Table 2 (16S971, 16S1228 or 16s1294) as shown in Table 4. The two mycoplasma species in the list (M. genitalium and M. pneumoniae) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced resolving power for closely related organisms. In cases such as Bacillus anthracis and Bacillus cereus that are virtually indistinguishable from each other based solely on mass differences, compositional analysis or fragmentation patterns are used to resolve the differences. The single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
  • [0096]
    Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background.
    TABLE 4a
    Organism name 23S_855 16S_1337 23S_1021
    Bacillus anthracis 42650.98 28447.65 30294.98
    Staphylococcus aureus 42654.97 28443.67 30297.96
  • [0097]
    [0097]
    TABLE 4b
    Organism name 16S_971 16S_1294 16S_1228
    Vibrio cholerae 55625.09 35856.87 52535.59
    Vibrio parahaemolyticus 54384.91 34620.67 50064.19
  • [0098]
    Table 4 shows the expected molecular weight and base composition of region 16S1100-1188 in Mycobacterium avium and Streptomyces sp.
    TABLE 5
    Organism Molecular
    Region name Length weight Base comp.
    16S Mycobacterium 82 25624.1728 A16G32C18T16
    1100-1188 avium
    16S Streptomyces 96 29904.871 A17G38C27T14
    1100-1188 sp.
  • [0099]
    Table 5 shows base composition (single strand) results for 16S1100-1188 primer amplification reactions different species of bacteria. Species which are repeated in the table (e.g., Clostridium botulinum) are different strains which have different base compositions in the 16S1100-1188 region.
    TABLE 6
    Organism name Base comp. Organism name Base comp.
    Mycobacterium avium A16G32C18T16 Vibrio cholerae A23G30C21T16
    Streptomyces sp. A17G38C27T14
    Figure US20040121314A1-20040624-P00810
    A 23 G 31 C 21 T 15
    Ureaplasma urealyticum A18G30C17T17
    Figure US20040121314A1-20040624-P00811
    A 23 G 31 C 21 T 15
    Streptomyces sp. A19G36C24T18 Mycoplasma genitalium A24G19C12T18
    Mycobacterium leprae A20G32C22T16 Clostridium botulinum A24G25C18T20
    Figure US20040121314A1-20040624-P00801
    A 20 G 33 C 21 T 16 Bordetella bronchiseptica A24G26C19T14
    Figure US20040121314A1-20040624-P00802
    A 20 G 33 C 21 T 16 Francisella tularensis A24G26C19T19
    Fusobacterium necroforum A21G26C22T16
    Figure US20040121314A1-20040624-P00805
    A 24 G 26 C 20 T 18
    Listeria monocytogenes A21G27C19T19
    Figure US20040121314A1-20040624-P00812
    A 24 G 26 C 20 T 18
    Clostridium botulinum A21G27C19T21
    Figure US20040121314A1-20040624-P00813
    A 24 G 26 C 20 T 18
    Neisseria gonorrhoeae A21G28C21T18 Helicobacter pylori A24G26C20T19
    Bartonella quintana A21G30C22T16 Helicobacter pylori A24G26C21T18
    Enterococcus faecalis A22G27C20T19 Moraxella catarrhalis A24G26C23T16
    Bacillus megaterium A22G28C20T18 Haemophilus influenzae Rd A24G28C20T17
    Bacillus subtilis A22G28C21T17
    Figure US20040121314A1-20040624-P00814
    A 24 G 28 C 21 T 16
    Pseudomonas aeruginosa A22G29C23T15
    Figure US20040121314A1-20040624-P00815
    A 24 G 28 C 21 T 16
    Legionella pneumophila A22G32C20T16
    Figure US20040121314A1-20040624-P00816
    A 24 G 28 C 21 T 16
    Mycoplasma pneumoniae A23G20C14T16 Pseudomonas putida A24G29C21T16
    Clostridium botulinum A23G26C20T19
    Figure US20040121314A1-20040624-P00817
    A 24 G 30 C 21 T 15
    Enterococcus faecium A23G26C21T18
    Figure US20040121314A1-20040624-P00818
    A 24 G 30 C 21 T 15
    Acinetobacter calcoaceti A23G26C21T19
    Figure US20040121314A1-20040624-P00819
    A 24 G 30 C 21 T 15
    Figure US20040121314A1-20040624-P00803
    A 23 G 26 C 24 T 15 Clostridium botulinum A25G24C18T21
    Figure US20040121314A1-20040624-P00804
    A 23 G 26 C 24 T 15 Clostridium tetani A25G25C18T20
    Clostridium perfringens A23G27C19T19 Francisella tularensis A25G25C19T19
    Figure US20040121314A1-20040624-P00805
    A 23 G 27 C 20 T 18 Acinetobacter calcoacetic A25G26C20T19
    Figure US20040121314A1-20040624-P00806
    A 23 G 27 C 20 T 18 Bacteriodes fragilis A25G27C16T22
    Figure US20040121314A1-20040624-P00807
    A 23 G 27 C 20 T 18 Chlamydophila psittaci A25G27C21T16
    Aeromonas hydrophila A23G29C21T16 Borrelia burgdorferi A25G29C17T19
    Escherichia coli A23G29C21T16 Streptobacillus monilifor A26G26C20T16
    Pseudomonas putida A23G29C21T17 Rickettsia prowazekii A26G28C18T18
    Figure US20040121314A1-20040624-P00808
    A 23 G 29 C 22 T 15 Rickettsia rickettsii A26G28C20T16
    Figure US20040121314A1-20040624-P00809
    A 23 G 29 C 22 T 15 Mycoplasma mycoides A28G23C16T20
  • [0100]
    The same organism having different base compositions are different strains. Groups of organisms which are highlighted or in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example, Bacillus anthracis can be distinguished from Bacillus cereus and Bacillus thuringiensis using the primer 16S971-1062 (Table 6). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g. anthracis cluster) are distinguished at high resolution with focused sets of primer pairs. The known biowarfare agents in Table 6 are Bacillus anthracis, Yersinia pestis, Francisella tularensis and Rickettsia prowazekii.
    TABLE 7
    Organism 16S_971-1062 16S_1228-1310 16S_1100-1188
    Aeromonas hydrophila A21G29C22T20 A22G27C21T13 A23G31C21T15
    Aeromonas salmonicida A21G29C22T20 A22G27C21T13 A23G31C21T15
    Bacillus anthracis A 21 G 27 C 22 T 22 A24G22C19T18 A23G27C20T18
    Bacillus cereus A22G27C21T22 A24G22C19T18 A23G27C20T18
    Bacillus thuringiensis A22G27C21T22 A24G22C19T18 A23G27C20T18
    Chlamydia trachomatis A 22 G 26 C 20 T 23 A 24 G 23 C 19 T 16 A24G28C21T16
    Chlamydia pneumoniae AR39 A26G23C20T22 A26G22C16T18 A24G28C21T16
    Leptospira borgpetersenii A22G26C20T21 A22G25C21T15 A23G26C24T15
    Leptospira interrogans A22G26C20T21 A22G25C21T15 A23G26C24T15
    Mycoplasma genitalium A28G23C15T22 A 30 G 18 C 15 T 19 A 24 G 19 C 12 T 18
    Mycoplasma pneumoniae A28G23C15T22 A 27 G 19 C 16 T 20 A 23 G 20 C 14 T 16
    Escherichia coli A 22 G 28 C 20 T 22 A24G25C21T13 A23G29C22T15
    Shigella dysenteriae A 22 G 28 C 21 T 21 A24G25C21T13 A23G29C22T15
    Proteus vulgaris A 23 G 26 C 22 T 21 A 26 G 24 C 19 T 14 A24G30C21T15
    Yersinia pestis A24G25C21T22 A25G24C20T14 A24G30C21T15
    Yersinia pseudotuberculosis A24G25C21T22 A25G24C20T14 A24G30C21T15
    Francisella tularensis A 20 G 25 C 21 T 23 A 23 G 26 C 17 T 17 A 24 G 26 C 19 T 19
    Rickettsia prowazekii A 21 G 26 C 24 T 25 A 24 G 23 C 16 T 19 A 26 G 28 C 18 T 18
    Rickettsia rickettsii A 21 G 26 C 25 T 24 A 24 G 24 C 17 T 17 A 26 G 28 C 20 T 16
  • [0101]
    [0101]
    B. anthracis_16S_971
    GCGAAGAACCUUACCAGGUNUUGACAUCCUCUGACAACCCUAGAGAUAGGGCU (SEQ ID NO: 1)
    UCUCCUUCGGGAGCAGAGUGACAGGUGGUGCAUGGUU
    B. cereus_16S_971
    GCGAAGAACCUUACCAGGUCUUGACAUCCUCUGAAAACCCUAGAGAUAGGGCU (SEQ ID NO: 2)
    UCUCCUUCGGGAGCAGAGUGACAGGUGGUGCAUGGUU
  • Example 6 ESI-TOF MS of sspE 56-mer Plus Calibrant
  • [0102]
    The mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in FIG. 8. The mass standard was a 20-mer phosphorothioate oligonucleotide added to a solution containing a 56-mer PCR product from the B. anthracis spore coat protein sspE. The mass of the expected PCR product distinguishes B. anthracis from other species of Bacillus such as B. thuringiensis and B. cereus.
  • Example 7 B. anthracis ESI-TOF Synthetic 16S1228 Duplex
  • [0103]
    An ESI-TOF MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1228 region of the B. anthracis 16S rRNA gene. The results (FIG. 9) show that the molecular weights of the forward and reverse strands can be accurately determined and easily distinguish the two strands. The [M-21H+]21− and [M-20H+]20− charge states are shown.
  • Example 8 ESI-FTICR-MS of Synthetic B. anthracis 16S1337 46 Base Pair Duplex
  • [0104]
    An ESI-FTICR-MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1337 region of the B. anthracis 16S rRNA gene. The results (FIG. 10) show that the molecular weights of the strands can be distinguished by this method. The [M-16H+]16− through [M-10H+]10− charge states are shown. The insert highlights the resolution that can be realized on the FTICR-MS instrument, which allows the charge state of the ion to be determined from the mass difference between peaks differing by a single 13C substitution.
  • Example 9 ESI-TOF MS of 56-mer Oligonucleotide from saspB Gene of B. anthracis with Internal Mass Standard
  • [0105]
    ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5μM )from the saspB gene of B. anthracis containing an internal mass standard at an ESI of 1.7 μL/min as a function of sample consumption. The results (FIG. 11) show that the signal to noise is improved as more scans are summed, and that the standard and the product are visible after only 100 scans.
  • Example 10 ESI-TOF MS of an Internal Standard with Tributylammonium (TBA)-trifluoroacetate (TFA) Buffer
  • [0106]
    An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standard was obtained following addition of 5 mM TBA-TFA buffer to the solution. This buffer strips charge from the oligonucleotide and shifts the most abundant charge state from [M-8H+]8− to [M-3H+]3− (FIG. 12).
  • [0107]
    Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.
  • 1 7 1 90 RNA Bacillus anthracis misc_feature (20)..(20) N = A, U, G or C 1 gcgaagaacc uuaccaggun uugacauccu cugacaaccc uagagauagg gcuucuccuu 60 cgggagcaga gugacaggug gugcaugguu 90 2 90 RNA Bacillus cereus 2 gcgaagaacc uuaccagguc uugacauccu cugaaaaccc uagagauagg gcuucuccuu 60 cgggagcaga gugacaggug gugcaugguu 90 3 1542 RNA Artificial Sequence misc_feature 16S rRNA consensus sequence 3 nnnnnnnaga guuugaucnu ggcucagnnn gaacgcuggc ggnnngcnun anacaugcaa 60 gucgancgnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agnggcnnac gggugaguaa 120 nncnunnnna nnunccnnnn nnnnnggnan annnnnnnga aannnnnnnu aauaccnnau 180 nnnnnnnnnn nnnnaaagnn nnnnnnnnnn nnnnnnnnnn nnnnnngann nnnnnnngnn 240 nnaunagnun guuggunngg uaanggcnna ccaagncnnn gannnnuagc ngnncugaga 300 ggnngnncng ccacanuggn acugaganac ggnccanacu ccuacgggag gcagcagunn 360 ggaaunuunn ncaauggnng naanncugan nnagcnannc cgcgugnnng anganggnnu 420 nnngnungua aannncunun nnnnnngang annnnnnnnn nnnnnnnnnn nnnnnnnnnu 480 gacnnuannn nnnnannaag nnncggcnaa cuncgugcca gcagccgcgg uaauacgnag 540 gnngcnagcg uunnncggan unanugggcg uaaagngnnn gnaggnggnn nnnnnngunn 600 nnngunaaan nnnnnngcun aacnnnnnnn nnncnnnnnn nacnnnnnnn cungagnnnn 660 nnagnggnnn nnngaauunn nnguguagng gugnaauncg naganaunng nangaanacc 720 nnungcgaag gcnnnnnncu ggnnnnnnac ugacncunan nnncgaaagc nugggnagcn 780 aacaggauua gauacccugg uaguccangc nnuaaacgnu gnnnnnunnn ngnnngnnnn 840 nnnnnnnnnn nnnnnnnnna nnnaacgnnn uaannnnncc gccuggggag uacgnncgca 900 agnnunaaac ucaaangaau ugacggggnc cngcacaagc ngnggagnau guggnuuaau 960 ucgangnnac gcgnanaacc uuaccnnnnn uugacaunnn nnnnnnnnnn nnganannnn 1020 nnnnnnnnnn nnnnnnnnnn nnnacaggug nugcauggnu gucgucagcu cgugnnguga 1080 gnuguugggu uaagucccgn aacgagcgca acccnnnnnn nnnguuncna ncnnnnnnnn 1140 ngngnacucn nnnnnnacug ccnnngnnaa nnnggaggaa ggnggggang acgucaanuc 1200 nucaugnccc uuangnnnng ggcuncacac nuncuacaau ggnnnnnaca nngngnngcn 1260 annnngnnan nnnnagcnaa ncnnnnaaan nnnnucnnag uncggaungn nnncugcaac 1320 ucgnnnncnu gaagnnggan ucgcuaguaa ucgnnnauca gnangnnncg gugaauacgu 1380 ucncgggncu uguacacacc gcccgucann ncangnnagn nnnnnnnncc nnaagnnnnn 1440 nnnnnnncnn nnnngnnnnn nnnnncnang gnnnnnnnnn nganugggnn naagucguaa 1500 caagguancc nuannngaan nugnggnugg aucaccuccu un 1542 4 2904 RNA Artificial Sequence misc_feature 23S rRNA consensus sequence 4 nnnnaagnnn nnaagngnnn nngguggaug ccunggcnnn nnnagncgan gaaggangnn 60 nnnnncnncn nnanncnnng gnnagnngnn nnnnnncnnn nnanccnnng nunuccgaau 120 ggggnaaccc nnnnnnnnnn nnnnnnnnan nnnnnnnnnn nnnnnnnnnn nnnnnnngnn 180 nacnnnnnga anugaaacau cunaguannn nnaggaanag aaannaannn ngauuncnnn 240 nguagnggcg agcgaannng nannagncnn nnnnnnnnnn nnnnnnnnnn nnnannngaa 300 nnnnnuggna agnnnnnnnn nannngguna nannccngua nnnnaaannn nnnnnnnnnn 360 nnnnnnnnnn aguannncnn nncncgngnn annnngunng aannngnnnn gaccannnnn 420 naagncuaaa uacunnnnnn ngaccnauag ngnannagua cngugangga aaggngaaaa 480 gnacccnnnn nangggagug aaanagnncc ugaaaccnnn nncnuanaan nngunnnagn 540 nnnnnnnnnn nnnuganngc gunccuuuug nannaugnnn cngnganuun nnnunnnnng 600 cnagnuuaan nnnnnnnngn agncgnagng aaancgagun nnaanngngc gnnnagunnn 660 nngnnnnaga cncgaancnn ngugancuan nnaugnncag gnugaagnnn nnguaanann 720 nnnuggaggn ccgaacnnnn nnnnguugaa aannnnnngg augannugug nnungnggng 780 aaanncnaan cnaacnnngn nauagcuggu ucucnncgaa annnnuuuag gnnnngcnun 840 nnnnnnnnnn nnnnggnggu agagcacugn nnnnnnnnng gnnnnnnnnn nnnnuacnna 900 nnnnnnnnaa acuncgaaun ccnnnnnnnn nnnnnnnngn agnnanncnn ngngngnuaa 960 nnuncnnngu nnanagggna acancccaga ncnncnnnua aggncccnaa nnnnnnnnua 1020 aguggnaaan gangugnnnn nncnnanaca nnnaggangu uggcuuagaa gcagccancn 1080 uunaaagann gcguaanagc ucacunnucn agnnnnnnng cgcngannau nuancgggnc 1140 uaannnnnnn nccgaannnn nngnnnnnnn nnnnnnnnnn nnnnngguag nngagcgunn 1200 nnnnnnnnnn ngaagnnnnn nngnnannnn nnnuggannn nnnnnnagug ngnaugnngn 1260 naunaguanc gannnnnnnn gugananncn nnnncnccgn annncnaagg nuuccnnnnn 1320 nangnunnuc nnnnnngggu nagucgnnnc cuaagnngag ncnganangn nuagnngaug 1380 gnnannnggu nnauauuccn nnacnnnnnn nnnnnnnnnn nnnnngacgn nnnnngnnnn 1440 nnnnnnnnnn nnnnggnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnncnngaa aannnnnnnn nnnnnnnnnn nnnnnnnnnc guaccnnaaa ccgacacagg 1620 ungnnnngnn gagnanncnn aggngnnngn nnnaannnnn nnnaaggaac unngcaaanu 1680 nnnnccguan cuucggnana aggnnnncnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnng nnnnannnan nngnnnnnnn cnacuguuua nnaaaaacac agnncnnugc 1800 naanncgnaa gnnganguau anggnnugac nccugcccng ugcnngaagg uuaanngnnn 1860 nnnnnngnnn nngnnnnnnn nnnnannnaa gcccnnguna acggcggnng uaacuauaac 1920 nnuccuaagg uagcgaaauu ccuugucggg uaaguuccga ccngcacgaa nggngnaang 1980 annnnnnnnc ugucucnnnn nnnnncncng ngaanuunna nunnnnguna agaugcnnnn 2040 uncncgcnnn nngacggaaa gaccccnngn ancuuuacun nannnunnna nugnnnnnnn 2100 nnnnnnnnug unnagnauag gunggagncn nngannnnnn nncgnnagnn nnnnnggagn 2160 cnnnnnugnn auacnacncu nnnnnnnnnn nnnnucuaac nnnnnnnnnn nancnnnnnn 2220 nnngacanug nnngnngggn aguuunacug gggcggunnc cuccnaaann guaacggagg 2280 ngnncnaagg unnncunann nnggnnggnn aucnnnnnnn nagunnaann gnanaagnnn 2340 gcnunacugn nagnnnnacn nnncgagcag nnncgaaagn nggnnnuagu gauccggngg 2400 unnnnnnugg aagngccnuc gcucaacgga uaaaagnuac ncnggggaua acaggcunau 2460 nnnncccaag aguncanauc gacggnnnng uuuggcaccu cgaugucggc ucnucncauc 2520 cuggggcugn agnngguccc aagggunngg cuguucgccn nuuaaagngg nacgngagcu 2580 ggguunanaa cgucgugaga caguungguc ccuaucngnn gngngngnnn gannnuugan 2640 nngnnnugnn cnuaguacga gaggaccggn nngnacnnan cncuggugnn ncnguugunn 2700 ngccannngc anngcngnnu agcuannunn ggnnnngaua anngcugaan gcaucuaagn 2760 nngaancnnn cnnnnagann agnnnucncn nnnnnnnnnn nnnnnnnnna gnnncnnnnn 2820 agannannnn gungauaggn nngnnnugna agnnnngnna nnnnunnagn nnacnnnuac 2880 uaaunnnncn nnnnncuunn nnnn 2904 5 13 DNA Artificial Sequence misc_feature Primer 5 cgtggtgacc ctt 13 6 14 DNA Artificial Sequence misc_feature Primer 6 cgtcgtcacc gcta 14 7 13 DNA Artificial Sequence misc_feature Primer 7 cgtggtaccc ctt 13

Claims (43)

    What is claimed is:
  1. 1. A method of identifying an unknown bioagent in a container comprising:
    a) contacting nucleic acid from said bioagent in said container with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of the bioagent;
    b) amplifying said variable nucleic acid sequence to produce an amplification product;
    c) determining the molecular mass of said amplification product; and
    d) comparing said molecular mass to one or more molecular masses of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein a match identifies said unknown bioagent in said container.
  2. 2. The method of claim 1 wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.
  3. 3. The method of claim 1 wherein said amplifying step comprises polymerase chain reaction.
  4. 4. The method of claim 1 wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.
  5. 5. The method of claim 1 wherein said bioagent is a bacterium, virus, parasite, fungi, cell or spore.
  6. 6. The method of claim 1 wherein said nucleic acid is ribosomal RNA.
  7. 7. The method of claim 1 wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
  8. 8. The method of claim 1 wherein said amplification product is ionized prior to molecular mass determination.
  9. 9. The method of claim 1 further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.
  10. 10. The method of claim 1 further comprising the step of performing steps a)-d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps a)-c) on a different plurality of known organisms from those in step d).
  11. 11. The method of claim 1 wherein said one or more molecular masses are contained in a database of molecular masses.
  12. 12. The method of claim 1 wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
  13. 13. The method of claim 1 wherein said molecular mass is determined by mass spectrometry.
  14. 14. The method of claim 11 wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.
  15. 15. The method of claim 1 further comprising performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
  16. 16. The method of claim 1 wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.
  17. 17. The method of claim 16 wherein said primer comprises a universal base at position 3 of each triplet within said primer.
  18. 18. The method of claim 16 wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.
  19. 19. The method of claim 16 wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
  20. 20. The method of claim 1 wherein said container is a package, box, envelope, mail tube, or railroad box car.
  21. 21. A method of identifying an unknown bioagent in a container comprising:
    a) contacting nucleic acid from said bioagent in a container with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence;
    b) amplifying said variable nucleic acid sequence to produce an amplification product;
    c) determining the base composition of said amplification product; and
    d) comparing said base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein a match identifies said unknown bioagent in said container.
  22. 22. The method of claim 21 wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.
  23. 23. The method of claim 21 wherein said amplifying step comprises polymerase chain reaction.
  24. 24. The method of claim 21 wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.
  25. 25. The method of claim 21 wherein said bioagent is a bacterium, virus, fungi, parasite, cell or spore.
  26. 26. The method of claim 21 wherein said nucleic acid is ribosomal RNA.
  27. 27. The method of claim 21 wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
  28. 28. The method of claim 21 wherein said amplification product is ionized prior to base composition determination.
  29. 29. The method of claim 21 further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.
  30. 30. The method of claim 21 further comprising the step of performing steps a)-d) using a different oligonucleotide primer pair and comparing the results to one or more base compositions of amplification product obtained by performing steps a)-c) on a different plurality of known organisms from those in step d).
  31. 31. The method of claim 21 wherein said one or more base composition signatures are contained in a database of base composition signatures.
  32. 32. The method of claim 21 wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
  33. 33. The method of claim 21 wherein said base composition signature is determined by mass spectrometry.
  34. 34. The method of claim 33 wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), q-TOF and triple quadrupole.
  35. 35. The method of claim 21 further comprising performing step b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
  36. 36. The method of claim 21 wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.
  37. 37. The method of claim 36 wherein said primer comprises a universal base at position 3 of each triplet within said primer.
  38. 38. The method of claim 36 wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.
  39. 39. The method of claim 36 wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
  40. 40. The method of claim 21 wherein said container is a package, box, envelope, mail tube, or railroad box car.
  41. 41. A method of determining the absence of a bioagent in a container sample comprising:
    a) contacting said container sample suspected of containing nucleic acid encoding said bioagent with at least one pair of oligonucleotide primers which are capable of hybridizing to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of said bioagent;
    b) treating said variable nucleic acid sequence under amplification conditions capable of producing an amplification product of said variable nucleic acid sequence;
    c) performing spectroscopy to determine the molecular mass or base composition of all amplification products; and
    d) comparing said molecular mass to one or more molecular masses of amplification products or said base composition to one or more base compositions of amplification products obtained by performing steps a)-c) on a plurality of known organisms, wherein the lack of a match indicates that said bioagent is absent from said container sample.
  42. 42. The method of claim 41 further comprising determining the presence of a positive control.
  43. 43. The method of claim 42 wherein said positive control is a known bioagent or residual primer signal.
US10326642 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents in containers Abandoned US20040121314A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US43131902 true 2002-12-06 2002-12-06
US32318602 true 2002-12-18 2002-12-18
US10326642 US20040121314A1 (en) 2002-12-06 2002-12-18 Methods for rapid detection and identification of bioagents in containers
US10326047 US20030190605A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents for environmental testing
US44378803 true 2003-01-30 2003-01-30
US45360703 true 2003-03-10 2003-03-10
US45416503 true 2003-03-12 2003-03-12
US10660996 US7255992B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents for environmental and product testing

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US10326642 US20040121314A1 (en) 2002-12-06 2002-12-18 Methods for rapid detection and identification of bioagents in containers
US10660996 US7255992B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents for environmental and product testing
PCT/US2003/038757 WO2004053076A3 (en) 2002-12-06 2003-12-05 Methods for rapid detection and identification of bioagents for environmental and product testing
US10808932 US20040253619A1 (en) 2002-12-06 2004-03-25 Methods for rapid detection and identification of bacterial bioagents
US10814754 US20040253583A1 (en) 2002-12-06 2004-03-31 Methods for rapid detection and identification of viral bioagents

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US10808932 Continuation US20040253619A1 (en) 2001-03-02 2004-03-25 Methods for rapid detection and identification of bacterial bioagents
US10814754 Continuation US20040253583A1 (en) 2001-03-02 2004-03-31 Methods for rapid detection and identification of viral bioagents

Publications (1)

Publication Number Publication Date
US20040121314A1 true true US20040121314A1 (en) 2004-06-24

Family

ID=32512824

Family Applications (4)

Application Number Title Priority Date Filing Date
US10326642 Abandoned US20040121314A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents in containers
US10660996 Active 2022-04-10 US7255992B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents for environmental and product testing
US10808932 Abandoned US20040253619A1 (en) 2001-03-02 2004-03-25 Methods for rapid detection and identification of bacterial bioagents
US10814754 Abandoned US20040253583A1 (en) 2001-03-02 2004-03-31 Methods for rapid detection and identification of viral bioagents

Family Applications After (3)

Application Number Title Priority Date Filing Date
US10660996 Active 2022-04-10 US7255992B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents for environmental and product testing
US10808932 Abandoned US20040253619A1 (en) 2001-03-02 2004-03-25 Methods for rapid detection and identification of bacterial bioagents
US10814754 Abandoned US20040253583A1 (en) 2001-03-02 2004-03-31 Methods for rapid detection and identification of viral bioagents

Country Status (2)

Country Link
US (4) US20040121314A1 (en)
WO (1) WO2004053076A3 (en)

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040219517A1 (en) * 2001-03-02 2004-11-04 Ecker David J. Methods for rapid identification of pathogens in humans and animals
US20050270191A1 (en) * 2004-05-24 2005-12-08 Isis Pharmaceuticals, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US20080227087A1 (en) * 2005-10-11 2008-09-18 Ann Huletsky Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx
US20090263809A1 (en) * 2008-03-20 2009-10-22 Zygem Corporation Limited Methods for Identification of Bioagents
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US7741036B2 (en) 2001-03-02 2010-06-22 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
WO2011047307A1 (en) 2009-10-15 2011-04-21 Ibis Biosciences, Inc. Multiple displacement amplification
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
WO2011112718A1 (en) 2010-03-10 2011-09-15 Ibis Biosciences, Inc. Production of single-stranded circular nucleic acid
US8026084B2 (en) 2005-07-21 2011-09-27 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
US8046171B2 (en) 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
US8071309B2 (en) 2002-12-06 2011-12-06 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses
US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8158936B2 (en) 2009-02-12 2012-04-17 Ibis Biosciences, Inc. Ionization probe assemblies
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US8182992B2 (en) 2005-03-03 2012-05-22 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US8298760B2 (en) 2001-06-26 2012-10-30 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
WO2013036603A1 (en) 2011-09-06 2013-03-14 Ibis Biosciences, Inc. Sample preparation methods
US8407010B2 (en) 2004-05-25 2013-03-26 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8550694B2 (en) 2008-09-16 2013-10-08 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods
US8563250B2 (en) 2001-03-02 2013-10-22 Ibis Biosciences, Inc. Methods for identifying bioagents
US8871471B2 (en) 2007-02-23 2014-10-28 Ibis Biosciences, Inc. Methods for rapid forensic DNA analysis
US8950604B2 (en) 2009-07-17 2015-02-10 Ibis Biosciences, Inc. Lift and mount apparatus
US9068017B2 (en) 2010-04-08 2015-06-30 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity
US9149473B2 (en) 2006-09-14 2015-10-06 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens
US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification
US9393564B2 (en) 2009-03-30 2016-07-19 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods
US9416409B2 (en) 2009-07-31 2016-08-16 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US9777335B2 (en) 2001-06-04 2017-10-03 Geneohm Sciences Canada Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus

Families Citing this family (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110166040A1 (en) * 1997-09-05 2011-07-07 Ibis Biosciences, Inc. Compositions for use in identification of strains of e. coli o157:h7
US20100129811A1 (en) * 2003-09-11 2010-05-27 Ibis Biosciences, Inc. Compositions for use in identification of pseudomonas aeruginosa
US20090291446A1 (en) * 2004-04-15 2009-11-26 Institute For Environmental Health, Inc. Method for confirming the presence of an analyte
WO2006044728A3 (en) * 2004-10-18 2007-09-07 Us Genomics Inc Methods for isolation of nucleic acids from prokaryotic spores
JP5162447B2 (en) * 2005-04-13 2013-03-13 アイビス バイオサイエンシズ インコーポレイティッド Composition used for the identification of adenovirus
US20070111234A1 (en) * 2005-09-12 2007-05-17 Christian Birkner Detection of biological DNA
KR100659261B1 (en) * 2006-02-07 2006-12-12 한국기초과학지원연구원 Tandem fourier transform ion cyclotron resonance mass spectrometer
US8088582B2 (en) 2006-04-06 2012-01-03 Ibis Biosciences, Inc. Compositions for the use in identification of fungi
WO2007133189A3 (en) * 2006-05-01 2009-04-16 Us Gov Health & Human Serv Methods and agents for detecting parechovirus
EP2029777B1 (en) * 2006-05-31 2017-03-08 Sequenom, Inc. Methods and compositions for the extraction of nucleic acid from a sample
CN101501251A (en) * 2006-06-16 2009-08-05 塞昆纳姆股份有限公司 Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample
CA2658105C (en) * 2006-08-01 2016-07-05 Gen-Probe Incorporated Methods of nonspecific target capture of nucleic acids
US8148511B2 (en) * 2006-09-28 2012-04-03 The University Of North Carolina At Chapel Hill Methods and compositions for the detection and quantification of E. coli and Enterococcus
US7902345B2 (en) 2006-12-05 2011-03-08 Sequenom, Inc. Detection and quantification of biomolecules using mass spectrometry
US8076104B2 (en) * 2007-01-25 2011-12-13 Rogan Peter K Rapid and comprehensive identification of prokaryotic organisms
EP2140031A4 (en) 2007-03-26 2011-04-20 Sequenom Inc Restriction endonuclease enhanced polymorphic sequence detection
US7811766B2 (en) * 2007-03-28 2010-10-12 Thinkvillage, Llc Genetic identification and validation of Echinacea species
US8527207B2 (en) * 2007-05-15 2013-09-03 Peter K. Rogan Accurate identification of organisms based on individual information content
US20100291544A1 (en) * 2007-05-25 2010-11-18 Ibis Biosciences, Inc. Compositions for use in identification of strains of hepatitis c virus
WO2009038840A4 (en) * 2007-06-14 2009-12-10 Ibis Biosciences, Inc. Compositions for use in identification of adventitious contaminant viruses
US20100136555A1 (en) * 2007-07-06 2010-06-03 The Arizona Board Of Regents, A Body Corporate Acting For And On Behalf Of Northern Arizona Univ. Burkholderia Pseudomallei Diagnostic Genetic Elements that Predict Mortality in Melioidosis
EP2195452B1 (en) 2007-08-29 2012-03-14 Sequenom, Inc. Methods and compositions for universal size-specific polymerase chain reaction
US20110097704A1 (en) * 2008-01-29 2011-04-28 Ibis Biosciences, Inc. Compositions for use in identification of picornaviruses
WO2009120808A3 (en) 2008-03-26 2010-03-04 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US9200329B2 (en) 2008-05-19 2015-12-01 Biofire Diagnostics, Llc Rapid epidemiologic typing of bacteria
WO2009155103A3 (en) * 2008-05-30 2010-03-18 Ibis Biosciences, Inc. Compositions for use in identification of tick-borne pathogens
WO2009151982A1 (en) * 2008-05-30 2009-12-17 Ibis Biosciences, Inc. Compositions for use in identification of francisella
US20110151437A1 (en) * 2008-06-02 2011-06-23 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US20110200985A1 (en) * 2008-10-02 2011-08-18 Rangarajan Sampath Compositions for use in identification of herpesviruses
WO2010039755A1 (en) * 2008-10-02 2010-04-08 Ibis Biosciences, Inc. Compositions for use in identification of members of the bacterial genus mycoplasma
WO2010039787A1 (en) * 2008-10-03 2010-04-08 Ibis Biosciences, Inc. Compositions for use in identification of clostridium difficile
WO2010039848A3 (en) * 2008-10-03 2010-06-24 Ibis Biosciences, Inc. Compositions for use in identification of streptococcus pneumoniae
WO2010039870A1 (en) * 2008-10-03 2010-04-08 Ibis Biosciences, Inc. Compositions for use in identification of neisseria, chlamydia, and/or chlamydophila bacteria
WO2010039775A1 (en) * 2008-10-03 2010-04-08 Ibis Biosciences, Inc. Compositions for use in identification of members of the bacterial class alphaproteobacter
US20110190170A1 (en) * 2008-10-03 2011-08-04 Ibis Biosciences, Inc. Compositions for use in identification of antibiotic-resistant bacteria
WO2010080616A1 (en) 2008-12-19 2010-07-15 Abbott Laboratories Molecular assay for diagnosis of malaria
WO2010071821A1 (en) * 2008-12-19 2010-06-24 Abbott Laboratories Diagnostic test for mutations in codons 12-13 of human k-ras
US9719083B2 (en) 2009-03-08 2017-08-01 Ibis Biosciences, Inc. Bioagent detection methods
EP2414545B1 (en) 2009-04-03 2017-01-11 Sequenom, Inc. Nucleic acid preparation compositions and methods
EP3098325A1 (en) 2009-08-06 2016-11-30 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection
US20110065111A1 (en) * 2009-08-31 2011-03-17 Ibis Biosciences, Inc. Compositions For Use In Genotyping Of Klebsiella Pneumoniae
WO2011103144A1 (en) * 2010-02-16 2011-08-25 President And Fellows Of Harvard College Methods and systems for detection of microbes
WO2011115840A3 (en) * 2010-03-14 2011-11-10 Ibis Biosciences, Inc. Parasite detection via endosymbiont detection
WO2012159089A1 (en) 2011-05-19 2012-11-22 Sequenom, Inc. Products and processes for multiplex nucleic acid identification
WO2013036187A8 (en) * 2011-09-07 2013-04-25 Alpha Biotech Ab Determination of bacterial infections of the genus rickettsia and possibly borrelia, in patients exhibiting symptoms of disease and being blood donors
CA2983224A1 (en) 2015-04-24 2016-10-27 Agena Bioscience, Inc. Multiplexed method for the identification and quantitation of minor alleles and polymorphisms

Citations (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6423966B1 (en) *
US5484908A (en) * 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5502177A (en) * 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5580733A (en) * 1991-01-31 1996-12-03 Wayne State University Vaporization and sequencing of nucleic acids
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules
US5645985A (en) * 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5686242A (en) * 1991-09-05 1997-11-11 Isis Pharmaceuticals, Inc. Determination of oligonucleotides for therapeutics, diagnostics and research reagents
US5700642A (en) * 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method
US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US5830653A (en) * 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines
US5830655A (en) * 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US5849492A (en) * 1994-02-28 1998-12-15 Phylogenetix Laboratories, Inc. Method for rapid identification of prokaryotic and eukaryotic organisms
US5864137A (en) * 1996-10-01 1999-01-26 Genetrace Systems, Inc. Mass spectrometer
US5869242A (en) * 1995-09-18 1999-02-09 Myriad Genetics, Inc. Mass spectrometry to assess DNA sequence polymorphisms
US5871697A (en) * 1995-10-24 1999-02-16 Curagen Corporation Method and apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing
US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators
US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification
US5965363A (en) * 1996-09-19 1999-10-12 Genetrace Systems Inc. Methods of preparing nucleic acids for mass spectrometric analysis
US5981176A (en) * 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences
US5994066A (en) * 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US6001564A (en) * 1994-09-12 1999-12-14 Infectio Diagnostic, Inc. Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US6007992A (en) * 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6028183A (en) * 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same
US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US6051378A (en) * 1996-03-04 2000-04-18 Genetrace Systems Inc. Methods of screening nucleic acids using mass spectrometry
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6090558A (en) * 1997-09-19 2000-07-18 Genetrace Systems, Inc. DNA typing by mass spectrometry with polymorphic DNA repeat markers
US6104028A (en) * 1998-05-29 2000-08-15 Genetrace Systems Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6153389A (en) * 1999-02-22 2000-11-28 Haarer; Brian K. DNA additives as a mechanism for unambiguously marking biological samples
US6218118B1 (en) * 1998-07-09 2001-04-17 Agilent Technologies, Inc. Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry
US6235476B1 (en) * 1996-08-20 2001-05-22 Dako A/S Process for detecting nucleic acids by mass determination
US6235480B1 (en) * 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6238927B1 (en) * 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences
US6238871B1 (en) * 1993-01-07 2001-05-29 Sequenom, Inc. DNA sequences by mass spectrometry
US6268146B1 (en) * 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6268129B1 (en) * 1995-03-03 2001-07-31 Imperial Cancer Research Technology Limited Method of nucleic acid analysis
US6270974B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6270973B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6277578B1 (en) * 1998-03-13 2001-08-21 Promega Corporation Deploymerization method for nucleic acid detection of an amplified nucleic acid target
US6312902B1 (en) * 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity
US6372424B1 (en) * 1995-08-30 2002-04-16 Third Wave Technologies, Inc Rapid detection and identification of pathogens
US20020045178A1 (en) * 2000-06-13 2002-04-18 The Trustees Of Boston University Use of nucleotide analogs in the analysis of oligonucleotide mixtures and in highly multiplexed nucleic acid sequencing
US6391551B1 (en) * 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6432651B1 (en) * 1998-07-10 2002-08-13 Cetek Corporation Method to detect and analyze tight-binding ligands in complex biological samples using capillary electrophoresis and mass spectrometry
US6436640B1 (en) * 1999-03-18 2002-08-20 Exiqon A/S Use of LNA in mass spectrometry
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US20020137057A1 (en) * 2000-07-27 2002-09-26 Wold Barbara J. Rapid, quantitative method for the mass spectrometric analysis of nucleic acids for gene expression and genotyping
US6458533B1 (en) * 1997-12-19 2002-10-01 High Throughput Genomics, Inc. High throughput assay system for monitoring ESTs
US20020150927A1 (en) * 1999-04-30 2002-10-17 Matray Tracy J. Methods for detecting a plurality of analytes by mass spectrometry
US6475736B1 (en) * 2000-05-23 2002-11-05 Variagenics, Inc. Methods for genetic analysis of DNA using biased amplification of polymorphic sites
US6479239B1 (en) * 1998-03-10 2002-11-12 Large Scale Biology Corporation Detection and characterization of microorganisms
US20030017487A1 (en) * 2001-06-06 2003-01-23 Pharmacogenetics, Ltd. Method for detecting single nucleotide polymorphisms (SNP'S) and point mutations
US20030039976A1 (en) * 2001-08-14 2003-02-27 Haff Lawrence A. Methods for base counting
US20030064483A1 (en) * 1993-09-03 2003-04-03 Duke University. Method of nucleic acid sequencing
US20030073112A1 (en) * 2000-01-13 2003-04-17 Jing Zhang Universal nucleic acid amplification system for nucleic acids in a sample
US6558902B1 (en) * 1998-05-07 2003-05-06 Sequenom, Inc. Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules
US6582916B1 (en) * 1998-07-13 2003-06-24 Aventis Research & Technologies Gmbh & Co. Kg Metal ion-binding mass markers for nucleic acids
US20030129589A1 (en) * 1996-11-06 2003-07-10 Hubert Koster Dna diagnostics based on mass spectrometry
US20030134312A1 (en) * 2001-11-15 2003-07-17 Whatman, Inc. Methods and materials for detecting genetic material
US20030148284A1 (en) * 2001-12-17 2003-08-07 Vision Todd J. Solid phase detection of nucleic acid molecules
US6613509B1 (en) * 1999-03-22 2003-09-02 Regents Of The University Of California Determination of base (nucleotide) composition in DNA oligomers by mass spectrometry
US20030175729A1 (en) * 1999-12-29 2003-09-18 Van Eijk Michael Josephus Theresia Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using aflp
US6682889B1 (en) * 2000-11-08 2004-01-27 Becton, Dickinson And Company Amplification and detection of organisms of the Chlamydiaceae family
US20040038206A1 (en) * 2001-03-14 2004-02-26 Jia Zhang Method for high throughput assay of genetic analysis

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5567587A (en) * 1983-01-10 1996-10-22 Gen-Probe Incorporated Method for detecting, the presence and amount of prokaryotic organisms using specific rRNA subsequences as probes
US5270030A (en) * 1988-12-29 1993-12-14 Bio-Technology General Corp. Fibrin binding domain polypeptide and method of producing
ES2152933T3 (en) * 1991-10-23 2001-02-16 Baylor College Medicine Determination of traces on bacterial strains using amplifying repetitive DNA sequences.
WO1997037041A3 (en) 1996-03-18 1997-12-04 Sequenom Inc Dna sequencing by mass spectrometry
CA2222793A1 (en) * 1995-06-07 1996-12-19 Commonwealth Scientific And Industrial Research Organisation Optimized minizymes and miniribozymes and uses thereof
GB9602028D0 (en) * 1996-02-01 1996-04-03 Amersham Int Plc Nucleoside analogues
WO1998012355A1 (en) 1996-09-19 1998-03-26 Genetrace Systems Methods of preparing nucleic acids for mass spectrometric analysis
WO1998054751A1 (en) 1997-05-30 1998-12-03 Genetrace Systems, Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6221587B1 (en) * 1998-05-12 2001-04-24 Isis Pharmceuticals, Inc. Identification of molecular interaction sites in RNA for novel drug discovery
US6468743B1 (en) * 1998-05-18 2002-10-22 Conagra Grocery Products Company PCR techniques for detecting microbial contaminants in foodstuffs

Patent Citations (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6423966B1 (en) *
US5580733A (en) * 1991-01-31 1996-12-03 Wayne State University Vaporization and sequencing of nucleic acids
US5686242A (en) * 1991-09-05 1997-11-11 Isis Pharmaceuticals, Inc. Determination of oligonucleotides for therapeutics, diagnostics and research reagents
US5645985A (en) * 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) * 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5830653A (en) * 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines
US5981176A (en) * 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US6225450B1 (en) * 1993-01-07 2001-05-01 Sequenom, Inc. DNA sequencing by mass spectrometry
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5691141A (en) * 1993-01-07 1997-11-25 Sequenom, Inc. DNA sequencing by mass spectrometry
US6238871B1 (en) * 1993-01-07 2001-05-29 Sequenom, Inc. DNA sequences by mass spectrometry
US5851765A (en) * 1993-03-19 1998-12-22 Sequenon, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5872003A (en) * 1993-03-19 1999-02-16 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method
US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
US20030064483A1 (en) * 1993-09-03 2003-04-03 Duke University. Method of nucleic acid sequencing
US5502177A (en) * 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5763588A (en) * 1993-09-17 1998-06-09 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6005096A (en) * 1993-09-17 1999-12-21 Gilead Sciences, Inc. Pyrimidine derivatives
US5849492A (en) * 1994-02-28 1998-12-15 Phylogenetix Laboratories, Inc. Method for rapid identification of prokaryotic and eukaryotic organisms
US6001564A (en) * 1994-09-12 1999-12-14 Infectio Diagnostic, Inc. Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US6268129B1 (en) * 1995-03-03 2001-07-31 Imperial Cancer Research Technology Limited Method of nucleic acid analysis
US6268144B1 (en) * 1995-03-17 2001-07-31 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6258538B1 (en) * 1995-03-17 2001-07-10 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6589485B2 (en) * 1995-03-17 2003-07-08 Sequenom, Inc. Solid support for mass spectrometry
US20020150903A1 (en) * 1995-03-17 2002-10-17 Hubert Koster Diagnostics based on mass spectrometry
US6197498B1 (en) * 1995-03-17 2001-03-06 Sequenom, Inc DNA diagnostics based on mass spectrometry
US6602662B1 (en) * 1995-03-17 2003-08-05 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6277573B1 (en) * 1995-03-17 2001-08-21 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6235478B1 (en) * 1995-03-17 2001-05-22 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6221601B1 (en) * 1995-03-17 2001-04-24 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6221605B1 (en) * 1995-03-17 2001-04-24 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6300076B1 (en) * 1995-03-17 2001-10-09 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules
US5830655A (en) * 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US5700642A (en) * 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US6372424B1 (en) * 1995-08-30 2002-04-16 Third Wave Technologies, Inc Rapid detection and identification of pathogens
US5994066A (en) * 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US5869242A (en) * 1995-09-18 1999-02-09 Myriad Genetics, Inc. Mass spectrometry to assess DNA sequence polymorphisms
US5871697A (en) * 1995-10-24 1999-02-16 Curagen Corporation Method and apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6623928B2 (en) * 1996-01-23 2003-09-23 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6051378A (en) * 1996-03-04 2000-04-18 Genetrace Systems Inc. Methods of screening nucleic acids using mass spectrometry
US6468748B1 (en) * 1996-03-04 2002-10-22 Sequenom, Inc. Methods of screening nucleic acids using volatile salts in mass spectrometry
US20030113745A1 (en) * 1996-03-04 2003-06-19 Monforte Joseph A. Methods of screening nucleic acids using mass spectrometry
US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification
US6235476B1 (en) * 1996-08-20 2001-05-22 Dako A/S Process for detecting nucleic acids by mass determination
US5965363A (en) * 1996-09-19 1999-10-12 Genetrace Systems Inc. Methods of preparing nucleic acids for mass spectrometric analysis
US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US6111251A (en) * 1996-09-19 2000-08-29 Sequenom, Inc. Method and apparatus for MALDI analysis
US6566055B1 (en) * 1996-09-19 2003-05-20 Sequenom, Inc. Methods of preparing nucleic acids for mass spectrometric analysis
US6423966B2 (en) * 1996-09-19 2002-07-23 Sequenom, Inc. Method and apparatus for maldi analysis
US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity
US5864137A (en) * 1996-10-01 1999-01-26 Genetrace Systems, Inc. Mass spectrometer
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US20030129589A1 (en) * 1996-11-06 2003-07-10 Hubert Koster Dna diagnostics based on mass spectrometry
US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators
US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US6090558A (en) * 1997-09-19 2000-07-18 Genetrace Systems, Inc. DNA typing by mass spectrometry with polymorphic DNA repeat markers
US6028183A (en) * 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same
US6007992A (en) * 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6458533B1 (en) * 1997-12-19 2002-10-01 High Throughput Genomics, Inc. High throughput assay system for monitoring ESTs
US6479239B1 (en) * 1998-03-10 2002-11-12 Large Scale Biology Corporation Detection and characterization of microorganisms
US20030194699A1 (en) * 1998-03-13 2003-10-16 Promega Corporation Multiplex method for nucleic acid detection
US6235480B1 (en) * 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6277578B1 (en) * 1998-03-13 2001-08-21 Promega Corporation Deploymerization method for nucleic acid detection of an amplified nucleic acid target
US6312902B1 (en) * 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6268146B1 (en) * 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6270974B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6391551B1 (en) * 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
US6270973B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6558902B1 (en) * 1998-05-07 2003-05-06 Sequenom, Inc. Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules
US6265716B1 (en) * 1998-05-29 2001-07-24 Genetrace Systems, Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6104028A (en) * 1998-05-29 2000-08-15 Genetrace Systems Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6218118B1 (en) * 1998-07-09 2001-04-17 Agilent Technologies, Inc. Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry
US6432651B1 (en) * 1998-07-10 2002-08-13 Cetek Corporation Method to detect and analyze tight-binding ligands in complex biological samples using capillary electrophoresis and mass spectrometry
US6582916B1 (en) * 1998-07-13 2003-06-24 Aventis Research & Technologies Gmbh & Co. Kg Metal ion-binding mass markers for nucleic acids
US6238927B1 (en) * 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences
US6153389A (en) * 1999-02-22 2000-11-28 Haarer; Brian K. DNA additives as a mechanism for unambiguously marking biological samples
US6436640B1 (en) * 1999-03-18 2002-08-20 Exiqon A/S Use of LNA in mass spectrometry
US6613509B1 (en) * 1999-03-22 2003-09-02 Regents Of The University Of California Determination of base (nucleotide) composition in DNA oligomers by mass spectrometry
US20020150927A1 (en) * 1999-04-30 2002-10-17 Matray Tracy J. Methods for detecting a plurality of analytes by mass spectrometry
US20030175729A1 (en) * 1999-12-29 2003-09-18 Van Eijk Michael Josephus Theresia Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using aflp
US20030073112A1 (en) * 2000-01-13 2003-04-17 Jing Zhang Universal nucleic acid amplification system for nucleic acids in a sample
US6475736B1 (en) * 2000-05-23 2002-11-05 Variagenics, Inc. Methods for genetic analysis of DNA using biased amplification of polymorphic sites
US20020045178A1 (en) * 2000-06-13 2002-04-18 The Trustees Of Boston University Use of nucleotide analogs in the analysis of oligonucleotide mixtures and in highly multiplexed nucleic acid sequencing
US20020137057A1 (en) * 2000-07-27 2002-09-26 Wold Barbara J. Rapid, quantitative method for the mass spectrometric analysis of nucleic acids for gene expression and genotyping
US6682889B1 (en) * 2000-11-08 2004-01-27 Becton, Dickinson And Company Amplification and detection of organisms of the Chlamydiaceae family
US20040038206A1 (en) * 2001-03-14 2004-02-26 Jia Zhang Method for high throughput assay of genetic analysis
US20030017487A1 (en) * 2001-06-06 2003-01-23 Pharmacogenetics, Ltd. Method for detecting single nucleotide polymorphisms (SNP'S) and point mutations
US20030039976A1 (en) * 2001-08-14 2003-02-27 Haff Lawrence A. Methods for base counting
US20030134312A1 (en) * 2001-11-15 2003-07-17 Whatman, Inc. Methods and materials for detecting genetic material
US20030148284A1 (en) * 2001-12-17 2003-08-07 Vision Todd J. Solid phase detection of nucleic acid molecules

Cited By (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8017358B2 (en) 2001-03-02 2011-09-13 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US8268565B2 (en) 2001-03-02 2012-09-18 Ibis Biosciences, Inc. Methods for identifying bioagents
US9752184B2 (en) 2001-03-02 2017-09-05 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US20040219517A1 (en) * 2001-03-02 2004-11-04 Ecker David J. Methods for rapid identification of pathogens in humans and animals
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US9416424B2 (en) 2001-03-02 2016-08-16 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8563250B2 (en) 2001-03-02 2013-10-22 Ibis Biosciences, Inc. Methods for identifying bioagents
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US7741036B2 (en) 2001-03-02 2010-06-22 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US7781162B2 (en) 2001-03-02 2010-08-24 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8802372B2 (en) 2001-03-02 2014-08-12 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US8815513B2 (en) 2001-03-02 2014-08-26 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents in epidemiological and forensic investigations
US8017743B2 (en) 2001-03-02 2011-09-13 Ibis Bioscience, Inc. Method for rapid detection and identification of bioagents
US8017322B2 (en) 2001-03-02 2011-09-13 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US8214154B2 (en) 2001-03-02 2012-07-03 Ibis Biosciences, Inc. Systems for rapid identification of pathogens in humans and animals
US8265878B2 (en) 2001-03-02 2012-09-11 Ibis Bioscience, Inc. Method for rapid detection and identification of bioagents
US9777335B2 (en) 2001-06-04 2017-10-03 Geneohm Sciences Canada Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus
US8298760B2 (en) 2001-06-26 2012-10-30 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8921047B2 (en) 2001-06-26 2014-12-30 Ibis Biosciences, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8380442B2 (en) 2001-06-26 2013-02-19 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
US8822156B2 (en) 2002-12-06 2014-09-02 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US9725771B2 (en) 2002-12-06 2017-08-08 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8071309B2 (en) 2002-12-06 2011-12-06 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8046171B2 (en) 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
US8476415B2 (en) 2003-05-13 2013-07-02 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8013142B2 (en) 2003-09-11 2011-09-06 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US9447462B2 (en) 2004-02-18 2016-09-20 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US8187814B2 (en) 2004-02-18 2012-05-29 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses
US8173957B2 (en) 2004-05-24 2012-05-08 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US20050270191A1 (en) * 2004-05-24 2005-12-08 Isis Pharmaceuticals, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US9449802B2 (en) 2004-05-24 2016-09-20 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US7714275B2 (en) 2004-05-24 2010-05-11 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US8987660B2 (en) 2004-05-24 2015-03-24 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US8407010B2 (en) 2004-05-25 2013-03-26 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA
US9873906B2 (en) 2004-07-14 2018-01-23 Ibis Biosciences, Inc. Methods for repairing degraded DNA
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
US8182992B2 (en) 2005-03-03 2012-05-22 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
US8551738B2 (en) 2005-07-21 2013-10-08 Ibis Biosciences, Inc. Systems and methods for rapid identification of nucleic acid variants
US8026084B2 (en) 2005-07-21 2011-09-27 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
US20080227087A1 (en) * 2005-10-11 2008-09-18 Ann Huletsky Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx
US9149473B2 (en) 2006-09-14 2015-10-06 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens
US8871471B2 (en) 2007-02-23 2014-10-28 Ibis Biosciences, Inc. Methods for rapid forensic DNA analysis
US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US20090263809A1 (en) * 2008-03-20 2009-10-22 Zygem Corporation Limited Methods for Identification of Bioagents
US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8550694B2 (en) 2008-09-16 2013-10-08 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods
US9023655B2 (en) 2008-09-16 2015-05-05 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US9027730B2 (en) 2008-09-16 2015-05-12 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US8252599B2 (en) 2008-09-16 2012-08-28 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8609430B2 (en) 2008-09-16 2013-12-17 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US8158936B2 (en) 2009-02-12 2012-04-17 Ibis Biosciences, Inc. Ionization probe assemblies
US8796617B2 (en) 2009-02-12 2014-08-05 Ibis Biosciences, Inc. Ionization probe assemblies
US9165740B2 (en) 2009-02-12 2015-10-20 Ibis Biosciences, Inc. Ionization probe assemblies
US9393564B2 (en) 2009-03-30 2016-07-19 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods
US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification
US8950604B2 (en) 2009-07-17 2015-02-10 Ibis Biosciences, Inc. Lift and mount apparatus
US9416409B2 (en) 2009-07-31 2016-08-16 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US9890408B2 (en) 2009-10-15 2018-02-13 Ibis Biosciences, Inc. Multiple displacement amplification
EP2957641A1 (en) 2009-10-15 2015-12-23 Ibis Biosciences, Inc. Multiple displacement amplification
WO2011047307A1 (en) 2009-10-15 2011-04-21 Ibis Biosciences, Inc. Multiple displacement amplification
EP3225695A1 (en) 2009-10-15 2017-10-04 Ibis Biosciences, Inc. Multiple displacement amplification
WO2011112718A1 (en) 2010-03-10 2011-09-15 Ibis Biosciences, Inc. Production of single-stranded circular nucleic acid
US9752173B2 (en) 2010-04-08 2017-09-05 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity
US9068017B2 (en) 2010-04-08 2015-06-30 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity
WO2013036603A1 (en) 2011-09-06 2013-03-14 Ibis Biosciences, Inc. Sample preparation methods
EP3170831A1 (en) 2011-09-06 2017-05-24 Ibis Biosciences, Inc. Sample preparation methods

Also Published As

Publication number Publication date Type
WO2004053076A2 (en) 2004-06-24 application
US20040180328A1 (en) 2004-09-16 application
US20040253583A1 (en) 2004-12-16 application
US20040253619A1 (en) 2004-12-16 application
WO2004053076A3 (en) 2004-09-02 application
US7255992B2 (en) 2007-08-14 grant

Similar Documents

Publication Publication Date Title
Reischl et al. Real-Time PCR Assay Targeting IS481of Bordetella pertussis and Molecular Basis for Detecting Bordetella holmesii
Ishoey et al. Genomic sequencing of single microbial cells from environmental samples
Scott et al. Pneumonia research to reduce childhood mortality in the developing world
Rothman et al. Detection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated polymerase chain reaction assay
Fenollar et al. Molecular detection of Coxiella burnetii in the sera of patients with Q fever endocarditis or vascular infection
Koren et al. Human oral, gut, and plaque microbiota in patients with atherosclerosis
US20050266397A1 (en) Methods for identification of coronaviruses
Van Gelder Applications of the polymerase chain reaction to diagnosis of ophthalmic disease
Sontakke et al. Use of broad range16S rDNA PCR in clinical microbiology
Zeaiter et al. Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum
US6605451B1 (en) Methods and devices for multiplexing amplification reactions
Fenollar et al. Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria
Spigelman et al. The use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in ancient skeletons
Fredricks et al. Application of polymerase chain reaction to the diagnosis of infectious diseases
US20050266411A1 (en) Methods for rapid forensic analysis of mitochondrial DNA
US20070218467A1 (en) Methods for rapid identification and quantitation of nucleic acid variants
Steer et al. Classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region
US7635566B2 (en) Methods and compositions for isolating nucleic acid sequence variants
Hofreiter et al. The future of ancient DNA: Technical advances and conceptual shifts
Costa et al. Real-time PCR for diagnosis and follow-up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes
Hoffmann et al. Monitoring of putative vectors of bluetongue virus serotype 8, Germany
Emanuel et al. Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler
Rudi et al. Development and application of new nucleic acid-based technologies for microbial community analyses in foods
Wilson et al. Identification of Ciprofloxacin-ResistantCampylobacter jejuni by Use of a Fluorogenic PCR Assay
WO2007086904A2 (en) Compositions for use in identification of adenoviruses

Legal Events

Date Code Title Description
AS Assignment

Owner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ECKER, DAVID J.;GRIFFEY, RICHARD H.;SAMPATH, RANGARAJAN;AND OTHERS;REEL/FRAME:014001/0255;SIGNING DATES FROM 20030212 TO 20030218

AS Assignment

Owner name: IBIS BIOSCIENCES, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ISIS PHARMACEUTICALS, INC.;REEL/FRAME:019691/0584

Effective date: 20070814

Owner name: IBIS BIOSCIENCES, INC.,CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ISIS PHARMACEUTICALS, INC.;REEL/FRAME:019691/0584

Effective date: 20070814