WO2009120183A2 - Système pour la détection d’un pathogène biologique et utilisation de celui-ci - Google Patents

Système pour la détection d’un pathogène biologique et utilisation de celui-ci Download PDF

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Publication number
WO2009120183A2
WO2009120183A2 PCT/US2008/014042 US2008014042W WO2009120183A2 WO 2009120183 A2 WO2009120183 A2 WO 2009120183A2 US 2008014042 W US2008014042 W US 2008014042W WO 2009120183 A2 WO2009120183 A2 WO 2009120183A2
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Prior art keywords
pathogen
sample
detection
polynucleotide
analyte
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PCT/US2008/014042
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English (en)
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WO2009120183A3 (fr
Inventor
Colin J. H. Brenan
Tom Morrison
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Biotrove, Inc.
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Priority to CN2008801270693A priority Critical patent/CN102015996A/zh
Priority to US12/809,568 priority patent/US20110251084A1/en
Priority to EP08873532A priority patent/EP2231851A4/fr
Publication of WO2009120183A2 publication Critical patent/WO2009120183A2/fr
Publication of WO2009120183A3 publication Critical patent/WO2009120183A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0642—Filling fluids into wells by specific techniques
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features

Definitions

  • the present invention features a system that provides for the rapid and sensitive detection of a target analyte, such as a human pathogen, in a sample, such as an environmental sample or a clinical specimen.
  • a target analyte such as a human pathogen
  • the invention provides a system for detecting a target analyte (e.g., a pathogen polynucleotide or polypeptide), the system containing a first module that provides for target specific amplification; a second module that contains a detector that detects the presence or absence of an analyte (e.g., one or more analytes, such as a polynucleotide and/or polypeptide); and a third module (that may be the same or different than the first module) containing means for target specific pre-amplification or amplification of a polynucleotide (e.g., a pathogen polynucleotide and/or polypeptide); and a detector that identifies the specific analyte.
  • a target analyte e.g., a pathogen polynucleotide or polypeptide
  • detection of an analyte by the first module indicates that a target analyte is present in the sample and failure to detect an analyte indicates a target analyte is not present in the sample.
  • the system detects or identifies 1-50 or 1-20 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50) specific analytes.
  • target specific amplification occurs in a tube, bead, well, plate, channel, tubing, through-hole, microarray, or on a substrate.
  • the first module contains means for temperature control.
  • the amplification is carried out in an array of through-holes.
  • the detector detects a target specific polynucleotide and/or polypeptide.
  • the polynucleotide is bacterial, viral, fungal, or other pathogen.
  • the pathogen is any one or more of Vibrio cholerae, Legionella pneumonia, Cryptosporidium parvum, Staphylococcus aureus, and Bacillus anthracis.
  • the detector detects a Bacillus anthracis spore.
  • the pathogen is a plant pathogen that is any one or more of Myrothecium roridum, Phytophthora spp, Phytophthora infestans, Agrobacterium turn, Meloidogyne hapla, Colletotrichum coc, and Cylindocladium spatif, Verticillium dahlia, Veriicillium albo-tric, Rhizoctonia solani AG 4-1 , Rhizoctonia solani AG 2-2, G_proteob, Erwinea carot,
  • Rhizoctonia solani AG 4-2, and Fusarium oxysporum are the same or different.
  • the invention provides a method for detecting a pathogen, where the method employs a system delineated herein.
  • the invention provides a method for detecting and identifying a pathogen, the method involving amplifying a target specific polynucleotide in a sample; detecting the target specific polynucleotide, where detection of the polynucleotide indicates that the presence or absence of a pathogen in the sample; and identifying the target specific polynucleotide, thereby detecting and identifying the pathogen.
  • the invention provides a method for detecting and identifying a pathogen, the method involving amplifying a target specific polynucleotide in a sample; detecting the target specific polynucleotide, where the detection identifies the presence or absence of a target analyte in the sample; amplifying or pre-amplifying the target specific polynucleotide; and identifying the target specific polynucleotide, thereby detecting and identifying the pathogen.
  • the analyte, polynucleotide, or polypeptide is any one or more of Vibrio cholerae, Legionella pneumonia, Cryptosporidium parvum, Staphylococcus aureus, Bacillus anthracis, Myrothecium roridum, Phytophthora spp, Phytophthora infestans, Agrobacterium turn, Meloidogyne hapla, Colletotrichum coc, and Cylindocladium spatif, Verticillium dahlia, Verticillium albo-tric, Rhizoctonia solani AG 4-1 , Rhizoctonia solani AG 2-2, Qjproteob, Erwinea carot, Rhizoctonia solani AG 4-2, and Fusarium oxysporum.
  • the method further involves detecting a pathogen polypeptide, for example, using an immunoassay.
  • the sample is an environmental sample, such as an air sample, water sample, or environmental swab.
  • the invention provides a network of detection systems, the network containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 detection systems delineated herein in communication with a computer processing unit.
  • a detection systems delineated herein provides input regarding detection or identification of a pathogen to a computer processing unit, for example, via wireless communication method, ethernet connection, WiFi, mobile phone network, radio waves, blue tooth, microwave, or infrared methods.
  • the invention provides a combinatorial method for identifying multiple pathogens, the method comprising amplifying a target specific polynucleotide in a sample; and detecting the target specific polynucleotide using at least two probes each having a distinct detectable moiety, where detection of at least two moieties identifies the target specific polynucleotide, thereby detecting and identifying the pathogen.
  • the amplification is carried out in three separate PCR reactions and three dyes are used to detect at least 27 different targets.
  • detection of an analyte by the first module or in a first stage indicates that a target analyte is present in the sample and failure to detect an analyte indicates a target analyte is not present in the sample.
  • the system or method fails to detect an analyte, no further analysis of the sample is required.
  • the system detects or identifies 1-50 or 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50) specific analytes (e.g., pathogen polynucleotides or polypeptides).
  • target specific amplification occurs in a tube, bead, well, plate, channel, tubing, through-hole, microarray, or on a substrate.
  • the system includes means for temperature control.
  • the amplification is carried out in an array of through-holes.
  • the detector detects a target specific polynucleotide and/or polypeptide.
  • the polynucleotide is bacterial, viral, fungal, or other pathogen.
  • the pathogen is any one or more of Vibrio cholerae, Legionella pneumonia, Cryptosporidium parvum, Staphylococcus aureus, and Bacillus anthracis.
  • the detector detects a Bacillus anthracis spore.
  • the pathogen is a plant pathogen that is any one or more of Myrothecium roridum, Phytophthora spp, Phytophthora infestans, Agrobacterium turn, Meloidogyne hapla, Colletotrichum coc, and Cylindocladium spatif, Verticillium dahlia, Verticillium albo-tric, Rhizoctonia solani AG 4-1, Rhizoctonia solani AG 2-2, G_proteob, Erwinea carot,
  • the first and third modules are the same or different.
  • analyte is meant any nucleic acid molecule, polypeptide, marker, or fragments thereof.
  • alteration is meant an increase or decrease.
  • An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 75%, 80%, 90%, or 100%.
  • amplify is meant to increase the number of copies of a molecule.
  • the polymerase chain reaction PCR is used to amplify nucleic acids.
  • binding is meant having a physicochemical affinity for a molecule. Binding is measured by any of the methods of the invention, e.g., hybridization of a detectable nucleic acid probe, such as a TaqMan based probe, or Pleiades based probe.
  • detection system is meant a set of one or more devices that provides for the detection and/or the identification of an analyte.
  • sample any material collected for analysis.
  • detector refers to identifying the presence, absence, or level of an analyte.
  • detector is meant a device that distinguishes a signal.
  • detectable is meant a moiety that renders an analyte detectable. Detection may be by any means known in the art, including but not limited to radiological, spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels that render an analyte detectable include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, absorbing dyes, autofluorescent molecules, electron-dense reagents, enzymes, biotin, digoxigenin, haptens, aptamers, heavy metal atoms (substitute into DNA) and quantum dots.
  • detection of an analyte or pathogen is meant identifying the presence or absence of an analyte or a pathogen in a sample.
  • identification of an analyte or pathogen is meant identifying the
  • module is meant a system component.
  • nucleic acid or oligonucleotide probe is meant a polynucleotide capable of binding to a target nucleic acid of complementary sequence. Typically, such binding is accomplished through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labeled with isotopes, for example, chromophores, lumiphores, chromogens, or indirectly labeled with biotin to which a streptavidin complex may later bind.
  • isotopes for example, chromophores, lumiphores, chromogens, or indirectly labeled with biotin to which a streptavidin complex may later bind.
  • hybridize pair to form a double-stranded molecule between complementary polynucleotide sequences.
  • marker is meant any protein or polynucleotide that is associated with a pathogen.
  • “Microarray” means a collection of nucleic acid molecules or polypeptides from one or more organisms arranged on a solid support (for example, a chip, plate, or bead). These nucleic acid molecules or polypeptides may be arranged in a grid where the location of each nucleic acid molecule or polypeptide remains fixed.
  • nucleic acid molecule is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof.
  • Platen is meant a device having a high-density array of holes for holding and/or analyzing a plurality of liquid samples, e.g., described in US Patent Nos. 6,716,629; 6,027,873; 6,306,578; or 6,436,632, all of which are herein incorporated by reference.
  • Primary set means a set of oligonucleotides that hybridize to one or more polynucleotides.
  • a primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.
  • reference is meant a standard or control condition.
  • the phrase "selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (for example, total cellular or library DNA or RNA).
  • target nucleic acid molecule or polypeptide is meant a nucleic acid molecule, polypeptide, or biomarker of the sample that is to be detected.
  • target specific amplification is meant amplification of a target polynucleotide that occurs preferentially relative to non-specific amplification.
  • Figure 1 is a schematic diagram of a system for detection of a target analyte.
  • Figure 2 is a schematic diagram showing details for Stage 1 analysis.
  • Figure 3 is a schematic diagram showing details of an alternative approach to Stage 1 analysis.
  • Figure 4 is a schematic diagram showing direct detection of Stage 1 in detail, where a semi-quantitative dye scan is used during amplification.
  • Figure 5 is a table showing how combinations of target specific primers and dye specific probes can be used for pathogen detection.
  • Figure 6 is a table showing how combinations of target specific primers and dye specific probes can be used to detect specific pathogens.
  • Figure 7 is a schematic diagram of a system for detection of a test analyte using multiple singleton PCR detection.
  • Figures 8A and 8B are a graph and a table showing that data obtained from Real-Time
  • PCR on the OpenArrayTM platform was highly reproducible.
  • the standard deviation calculated across over 9000 data points was 0.1 ICt for 1000 target copies/through-hole, and 0.21Ct for 100 copies/through-hole.
  • Figures 9A-9C show a work flow scheme for pathogen detection, the anticipated performance of the detection system, and the anticipated benefits of the detection methods, respectively.
  • Figures 1 OA and 1 OB includes three graphs and a table that provide a quantitative analysis of Vibrio par ahaemolyticus, which was identified from a mix of 17 pathogens..
  • Figure 11 includes four graphs showing detection of targeted regions of Vibrio cholerae, Legionella pneumonia, Cryptosporidium parvum and Staphylococcus aureus DNA samples. Real time PCR is performed using primer pairs for each of these four organisms.
  • the X axis shows PCR cycle number.
  • the Y axis shows fluorescence.
  • NTC denotes no template control.
  • This Figure is an example of a Stage 2 detection step following amplification. This figure shows that pathogen detection occurs in the open array.
  • These graphs also shows the detection of specific amplification for each PCR product occurs in the presence of a mixture of pathogens.
  • Figure 12 is a schematic diagram showing schematic PRI-lock principle for the detection of one target.
  • Figure 13 is a schematic diagram showing an overview of the PRI-lock principle combined with the OpenArray technology for multiplex detection of three different targets
  • Figure 14 shows an image of real - time detection and quantification of a single target in serial dilutions within an OpenArray slide.
  • Figure 15 shows real - time multiplex detection and quantification of 9 different plant pathogens in an OpenArray slide.
  • Panel A shows the total number of used PRI-locks, targets in yellow were applied to the sample.
  • Panel B shows the Spotting pattern of the through-hole subarray.
  • Panel C shows a subarray picture after 32 cycles.
  • Panel D shows Ct values for the found targets.
  • the following pathogens were assayed: Myrothecium roridum, Phytophthora spp, Phytophthora infestans, Agrobacterium turn, Meloidogyne hapla, Colletotrichum coc, and Cylindocladium spatif, Verticillium dahlia, Verticillium albo-tric, Rhizoctonia solani AG 4-1, Rhizoctonia solani AG 2-2, G_proteob, Erwinea carot, Rhizoctonia solani AG 4-2, and Fusarium oxysporum.
  • the invention features systems, methods and compositions for the detection and identification of a target analyte (e.g., polynucleotide, polypeptide) in a sample.
  • a target analyte e.g., polynucleotide, polypeptide
  • the invention is based, at least in part, on the discovery that a system for DNA extraction and concentration from a sample (e.g., environmental sample, air sample, water sample) may be coupled to a microfluidic system for the specific amplification and detection of one or more polynucleotides of interest (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 75, 100).
  • the system of the invention employs a two-stage strategy for identification of an analyte (e.g., identification of a pathogen polynucleotide). The first stage involves detection of the presence or absence of an analyte in a sample.
  • This detection step is performed not to identify the analyte, but to determine if an analyte is present in the sample.
  • the signal produced from the first detection step may be generated by the presence of one or more analytes in the sample.
  • the second stage involves identification of the analyte.
  • the system is particularly advantageous for the rapid low cost identification of even minute quantities of polynucleotide in a sample.
  • the detection system provides for the identification of a pathogen polynucleotide or other analyte indicative of a biological threat.
  • the advantages of this two stage approach to target detection is multi-fold, particularly in the context of continuous monitoring of the environment for specific biological agents or for the rapid processing of a large number of clinical samples in the event of exposure to a contagious biological agent.
  • speed, accuracy and sensitivity of detection are key requirements for the detection system.
  • the rapidity of detection system identification of a threat i.e., the presence of a pathogen in a sample
  • identification of a pathogen will depend on the specific application, but generally identification of a pathogen in less than about two hours is desirable.
  • detection of the presence of a pathogen, and identification of the pathogen is accomplished in a time less than the physiological response to the infectious agent. Depending on the organism, this can be less than about 90 minutes, 60 minutes, or even 30 minutes.
  • Sensitivity of the detection system to different micro-organisms has to be sufficient to detect an increase in the presence of the pathogen relative to the level of pathogens typically present in the environment.
  • the detection system provides for detection of a pathogen (e.g., micro-organism) at a level relative to the level of pathogen typically present in the environment.
  • the present system advantageously provides for few false positive and false negative identifications, typically less than about l : 10 5 , 1 : 10 6 , 1 : 10 7 , 1 : 10 8 to ensure the agent is reliably detected in the environmental sample or in the clinical specimen.
  • an internal positive control is included to decrease the false positive rate.
  • a detection system of the invention comprises a first component or Stage 1 that provides for target specific amplification (or pre-amplif ⁇ cation) and a second component that comprises a detector ( Figure 1).
  • a schematic diagram describing the work flow for pathogen detection is shown in Figure 9A.
  • the target specific amplification may be carried out in one or a series of tubes, wells, plates, channels, tubings, microarrays, through-holes or any other container or substrate that provides a suitable reaction environment.
  • the amplification is carried out in an array of tubes, wells, or through-holes.
  • the first component further comprises means for temperature control or temperature cycling to facilitate an amplification reaction.
  • a temperature gradient may be imposed across one or two-dimensions of an array fabricated from thermally conductive material by holding at least one edge of the array at a specific temperature (e.g., creating a hot zone) ( Figure 2). If the temperature is changed with time, then the temperature distribution across the array can be varied. The rate of temperature change is generally proportional to the applied temperature and to the distance from the heat source. Temperature can be modified using any means known in the art, including but not limited to a peltier unit ( Figure 3), a water bath, ohmic heating of an electrically conductive plate or absorption of electromagnetic energy.
  • Stage 1 If the Stage 1 registers a positive result (i.e., indicates the presence of an analyte, such as a pathogen), the remaining sample is directed to the second stage for detection of the specific analyte(s) responsible for the positive signal observed in Stage 1.
  • the target may be pre-amplified using one or more pairs of target-specific primers, thereby generating one or more target-specific amplicons.
  • the amplicons are then directed to one or more individual reaction containers (e.g., an array of microwells or through-holes) comprising one or more pairs of target specific primers and one or more probes.
  • At least one, two, three, four, or five targets is assayed per pathogen. In other embodiments of Stage 2, at least about three or more replicates is assayed per threat. Internal positive and negative controls reduce false positive/negative rates in Stage 2. Alternatively, if a detectable amount of analyte is present following Stage 1 , the analyte is identified, for example, by specifically binding a detectable probe or antibody. After the amplified sample is introduced into a container, the container is fluidically sealed and thermally cycled. The detection may be carried out in multiple singleton PCR detections ( Figure 7).
  • each reaction is monitored simultaneously in real-time for detection of an amplicon product for quantification of each target in the sample. Quantification is accomplished relative to a reference to internal controls.
  • the array may include one or more of the following: o positive controls to establish that the assay(s) worked; o negative controls to identify the presence of a false positive result; o two, three, four or more replicates to increase the precision, yield and/or accuracy of the result; and, o multiple targets per pathogen may be detected to increase the specificity of the detection and to reduce the detection of false positives.
  • the sample from Stage 1 is divided into an array for detection of individual amplicons ( Figure 1).
  • the invention features a microfluidic circuit incorporating two detection stages, Stage 1 and Stage 2, where a test sample is split into two parts. One volume is directed for analysis in Stage 1 and if found to contain one or more analytes based on a positive signal, the second volume is directed for analysis in Stage 2 to identify the specific analytes responsible for the positive signal.
  • Stage 1 analysis is carried out using a continuous process.
  • Continuous-flow PCR chips may be manufactured in silicon, glass, disposable polymer microfluidic chips (e.g., polycarbonate) or metal. Such chips are microstructured using standard photolithography with a positive photoresist SU-8 and application of etching processes known in the art fabricate the microstructure pattern. Alternatively, the microstructure is derived by stamping the substrate with a stamp fabricated by subsequent electroplating of the developed resist structure with nickel.
  • the channel dimensions may vary, however, in one embodiment the channel is 500 ⁇ m width, 100 ⁇ m depth with an overall channel length of 818 mm.
  • the liquid After the inlet port, the liquid passes through a longer section of the microchannel at the denaturation temperature.
  • the chip then contains some number (e.g., 5, 10, 15, 20, 30, 50 or more) temperature cycles that are effected by the passage of the sample through a channel that passes through one, two, three or more temperature zones before exiting the chip after a post -elongation period.
  • the channel passes through a denaturing zone, an annealing zone and an elongation zone.
  • the total length per PCR cycle is between 35 and 74 mm (e.g., 35, 40, 50, 60, 70, 80, 90, 100 mm), which represents a microliter sample volume that undergoes thermal cycling as compared to a liquid volume of 200 ⁇ l in a typical PCR plate.
  • the continuous flow PCR is carried out on a disposable polymer disk having a long spiral microfluidic channel of varying width.
  • the disc may be sandwiched with heat blocks of constant temperature (Chung et al., "Continuous-flow PCR using a disposable polymer disk (www-samlab.unine.ch/ConferenceCD/IMCSl 1/pdfs/AP 190M.pdf).
  • the sample is monitored by one or more detectors as it traverses the channel or channels. This provides for the quantification of the amplicon as it accumulates overtime.
  • Real-time PCR approaches are based upon a change in fluorescence associated with the accumulation of amplification products.
  • the change in fluorescence is monitored in real time during thermal cycling. Fluorescence changes may be attributed to probe cleavage (e.g., TaqMan® chemistry), doublestranded DNA-binding dyes (e.g., SYBR® Green), primer extension (e.g., Molecular Beacons) or by incorporation of a fluorescence quencher to reduce the signal generated by a fluorescently-labeled primer (e.g., PlexorTM technology).
  • PLEXORTM Technology is used to detect product accumulation over time, which is measured as a reduction in fluorescent signal.
  • product accumulation is measured as a reduction in fluorescent signal during amplification.
  • the reaction uses only two primers, one of which contains both a fluorescent tag and a modified base. The other primer is unmodified.
  • fluorescence is reduced by the site-specific incorporation of a fluorescent quencher, which is attached to a modified nucleotide (iso-dG) and inserted opposite the complementary modified base (iso-dC).
  • the quencher is in close proximity to a fluorescent dye located on the 5' end of the primer, resulting in a reduction in the fluorescent signal.
  • the Stage 1 detects the presence of a pathogen using, for example, multiplex PCR.
  • Stage 2 one or more specific amplification products is used to identify the pathogen.
  • Stage 2 is carried out using batch processing for identification and quantification of positive threats in sample. To enhance specificity, two or more primer pairs may be used for each pathogen. Detection of each amplicon is then accomplished using specific fluorescent dyes. The detection of the specific combination of dyes indicates the positive identification of a pathogen in the sample. A positive control may be used to confirm that the assay worked. Failure of the assay could be associated with the presence of an inhibitor that interferes with PCR, fluidic error, reagent failure, or another problem.
  • the number of pathogen identification per PCR reaction is limited by the detection methods employed in a thermal cycler reaction vessel. Melting curve analysis allows discrimination of PCR products based on the thermal melting properties of the PCR products and may be used to identify specific PCR products. Alternatively, fluorescent dyes are coupled to specific PCR events. These methods are commonly practiced and allow a single PCR reaction detect multiple pathogens. The number of pathogens identified by these detection methods is limited by the resolution of the detector. For example, the spectral separation of fluorescent dyes limits the number of specific probes used in a single reaction, or the thermal separation of melting products limits the number of uniquely identifiable PCR products.
  • a simple solution is to run multiple PCR reactions, thereby multiplying the number of detectible targets by the number of additional PCR.
  • the approach in Figures 5 and 6 uses a combinatorial approach that extends the number of detectable targets beyond a simple multiplicity.
  • three PCR and three dyes are used to detect up to 27 different targets. Each target would require amplification of three unique probes in a specific PCR reaction vessel before the target is considered present.
  • an assay for 27 pathogens with three targets per pathogen would require 27 independent PCR using three different fluorescent dyes, instead of the three PCR reactions described by the approach depicted in Figure 5.
  • such methods use less sample than conventional methods.
  • the present invention provides for multiple (2, 3, 4, 5, 6 or more) reactions to be carried out and analysed in one step.
  • the sample is split into multiple separate reactions. If desired, the sample may be pre- amplif ⁇ ed to generate enough sample to preserve sensitivity.
  • Figure 5 The approach described in Figure 5 is particularly useful when the pathogen detected is rarely present, and multiple pathogens are unlikely to be identified in a single sample.
  • Figure 6 describes an alternative combinatorial approach that is particularly useful when multiple pathogens are likely to be present in a single sample. In this case three dyes and three independent PCR reactions resolve any combination of up to six pathogens (i.e., 1, 2, 3, 4, 5, 6).
  • This combinatorial approach can be incorporated with detection methods other than fluorescent dyes, such as melting curve profiles, capillary electrophoresis mobility, and charge to mass measured by mass spectrophotometer.
  • the output of the first stage may or may not be sufficient to unequivocally identify the pathogen. If sufficient amounts of polynucleotide are present, no additional amplification need be carried out. Instead, the polynucleotide from Stage 1 can be directly identified.
  • the first stage dye set is designed to signal the presence of bacteria, viruses or fungi in the specimen a different fluorescent probe is associated with the probe sequence targeting each class of micro-organism.
  • the same fluorescent probe or an intercalating dye such as SYBR Green I could be used with all primer sets and a generalized increase in fluorescent signal from the multiple reactions indicates the presence of a targeted micro-organism but does not identify the specific pathogenic agent.
  • a reaction container comprises a plurality of reaction vessels (e.g., wells, through-holes) to accommodate the detection of a plurality of pathogens (e.g., greater than 1 , 5, 10) simultaneously.
  • the geometrical configuration of the array provides for rapid thermal cycling. In one embodiment, less than about one hundred twenty, sixty minutes, forty-five minutes, thirty minutes, twenty minutes, or fifteen minutes is required for detection of a positive or negative signal.
  • a positive or negative signal is detected in at least about 15 cycles, 20 cycles, 25, 26, 27, 28, 29, or 30 cycles. In another embodiment a positive or negative signal is detected in at least about 30, 35, 40, 45, 50, 55, or 60 cycles.
  • nested primers are used in Stage 2 to increase the specificity of analyte detection and/or the specificity of PCR amplification.
  • the abundance of a target sequence is increased in the pre-amplification stage by PCR using target specific primers.
  • the primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids in the target locus.
  • the term "primer” as used herein refers to a sequence comprising two or more nucleobases.
  • the primer comprises 3, and most preferably more than 8 nucleobases, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a polymorphic locus strand.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization.
  • the exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition.
  • the oligonucleotide primer typically contains between 12 and 27 or more nucleotides, although it may contain fewer nucleotides (e.g., 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40).
  • Primers of the invention are designed to be "substantially" complementary to each strand of the genomic locus to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' flanking sequences to hybridize therewith and permit amplification of the genomic locus.
  • the amplified mixture of amplicons from stage 1 could be introduced directly into stage 2 and dispensed in the multi-container structure for individual and discrete detection of specific DNA or RNA targets in each distinct reaction container.
  • the detector identifies the presence or absence of an analyte (e.g., pathogen polynucleotide) in the sample.
  • the detector may distinguish any detectable signal in recognizing the analyte.
  • a detector may employ spectroscopic, photochemical, biochemical, immunochemical, or chemical means to detect the presence of the analyte.
  • the detection system includes a processing unit that receives input from the detector regarding the presence or absence of the analyte.
  • a PCR product i.e., amplicon
  • real-time PCR product is detected by probe binding.
  • probe binding generates a fluorescent signal, for example, by coupling a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates (e.g., TaqMan® (Applied Biosystems, Foster City, CA, USA), Pleiades (Nanogen, Inc., Bothell, WA, USA), Molecular Beacons (see, for example, Tyagi et al., Nature Biotechnology 14(3):303-8, 1996), Scorpions® (Molecular Probes Inc., Eugene, OR, USA)).
  • a PCR product is detected by the binding of a fluorogenic dye that emits a fluorescent signal upon binding (e.g., SYBR® Green (Molecular Probes)).
  • SYBR® Green Molecular Probes
  • each reaction container comprises one or more pair of primers, each of which is complementary to a polynucleotide of interest, and capable of amplifying that sequence.
  • the reaction container further comprises one or more probes that are detectably labeled.
  • the detectable probes each comprises a distinct fluorometric dye that provides for the separate detection of amplified target.
  • a microfluidic device or platen is in communication with an upstream sample collection device (e.g., an aerosol or liquid sampler).
  • the microfluidic device or platen is in communication with a downstream detector, i.e., an analytical device or means for carrying out analysis (e.g., mass spectroscopy (MS), nuclear magnetic resonance (NMR), capillary array electrophoresis (CAE), reverse transcription-PCR (RT-PCR), single molecule detection system, fluorescence detection, optical detection).
  • a downstream detector i.e., an analytical device or means for carrying out analysis (e.g., mass spectroscopy (MS), nuclear magnetic resonance (NMR), capillary array electrophoresis (CAE), reverse transcription-PCR (RT-PCR), single molecule detection system, fluorescence detection, optical detection).
  • Primer based fluorescent probes used for the primary detection step include but are not limited to LUX, which is commercially available from Invitrogen, and Plexor.
  • LUX denotes Light- Upon-Extension, which refers to the probe's characteristic increase in fluorescent intensity upon incorporation in double stranded DNA.
  • any method known in the art may be used for detection of an analyte.
  • one or more analytical methodologies can be performed on a through-hole, channel, reservoir, reaction chamber, capillary or combinations thereof.
  • the methods of the invention are particularly useful for monitoring for the presence of a pathogen in an environmental sample, such as an air sample, a water sample, an agricultural sample, or a biological sample.
  • the detection system described herein is coupled with a U.S. Genomics air sampling micro fluidic system for DNA extraction and concentration to address the needs of the biodefense community.
  • detection systems of the invention are deployed in a detection network.
  • the network monitors for pathogen detection over an area.
  • the network provides information on when a pathogen was first detected by a first detection system, whether the pathogen reached a second or third detection system, and the time that it took the pathogen to reach the next detector. This allows information to be gathered concerning, for example, the size, movement, and speed with which a pathogen or pathogen cloud is passing through an area.
  • the network comprises detection systems that transmits information from a detection system to one or more central computers for processing.
  • Test analytes e.g., polynucleotides, such as DNA, RNA
  • the second stage provides for the identification of the analyte in the sample indicates the analyte.
  • Detection of the presence of the target in the Stage 1 indicates that the sample should be further processed in aStage 2 identification process.
  • the Stage 2 increases the specificity and sensitivity of the assay, and definitively identifies the pathogen in the sample.
  • the present disclosure provides integrated modular systems for the preparation and analysis of target analytes from various samples.
  • the systems are useful in the preparation and analysis of various target analytes, including but not limited to, molecules (e.g. toxins, such as Ricin, or pharmaceuticals), biomolecules (e.g., polynucleotides, polypeptides, lipids), cells (e.g., eukaryotic and prokaryotic cells, such as bacteria), spores (e.g., B. anthracis), viruses (e.g., influenza, smallpox,), and other materials.
  • micro fluidic sample preparation and analysis can be performed by one or more of the system modules, as described herein.
  • a first module for purifying, or concentrating a target analyte include one or more of the following methods, including but not limited to lysis, cmulsification, sonication, centrifugation, chromatography, Solid Phase Extraction (SPE), immunocapture (e.g., immunomagnetic separations (IMS)), bead-based capture, and combinations thereof.
  • the first module can reduce a macroscale sample solution to a microscale volume, for example, by concentrating milliliters to microliters or smaller volumes for introduction into one or more microfiuidic devices, such as a platen comprising a through-hole.
  • a "microfiuidic device” as used herein refers to a device suitable for manipulating, storing, processing, or analyzing sub-milliliter quantities of fluid, such as microliter ( ⁇ L), nanoliter (nL), and/or picoliter (pL) volumes.
  • a microfiuidic device can comprise one or more microchips (e.g., micro-scale, nano-scale, pico-scale devices), capillaries or platens comprising through-holes.
  • the invention employs a microfiuidic or nanofluidic system that comprises a high density array of reaction containers, such as micro or nanoliter-scale through-holes, channels, or chambers for implementing a number of PCR analyses in less than about a 10, 20, or 100 microliters of fluid.
  • the invention employs the BioTrove nanofluidic system — a high density array of nanoliter-scale through- holes or chambers for implementing up to 3072 PCR analyses with 33 nl per reaction on an array the size of a microscope slide.
  • Such arrays are described, for example, by U.S. Patent No. 6,716,629, which is incorporated herein by reference.
  • the OpenArray (R) plate is a steel platen that comprises 3072 through holes having a diameter of about 320 ⁇ m. Each of the through holes is treated with a polymer to make the inside surface of each hole hydrophilic and the exterior surface hydrophobic. Liquid is dispensed and retained in each through-hole by means of surface force differentials between the liquid surface tension and the polymer coatings.
  • the through holes are grouped in forty-eight subarrays of sixty- four through holes each. The spacing between each subarray is about 4.5 mm.
  • the invention provides a platen comprising a high density array of nanoliter-scale through-holes or chambers comprising less than about a 1000 nl, 750 nl, 500 nl, 250 nl, 100 nl, or even 50 nl of the reagents and samples for PCR analyses.
  • Methods for loading the array with a small volume of reagents are described, for example, in U.S. Patent No. 6,716,629, 6,812,030, and 6,716,629, and in U.S. Patent Publication Nos. 200801081 12, 20030180807, and 20030124716.
  • the hydrophobic exterior surface of the platen is not wetted, keeping the liquid in each through-hole isolated from its neighbor.
  • PCR arrays are preloaded with PCR primers and probes.
  • Such reagents are typically transferred from 384- well plates into the through-holes with an array of 48 pins manipulated by a 4-axis robot, such that each through-hole of an OpenArray (R) plate has a different primer set.
  • the solvent is then removed resulting in the primers or primer/probes being immobilized on the inside surface of each hole.
  • Co-loading of a passive fluorescent reference dye allows detection of holes that failed to load assay.
  • the arrays are readily configurable as the assay configuration is based on the 384-well source plate layout.
  • the 3072 holes of the OpenArray (R) plate may be configured based on analytical needs; for example a sample can be interrogated by 16, 32, 64, multiples of 64, up to 3072 assays.
  • the systems disclosed herein have widespread applications in biodefense monitoring, infectious disease diagnostics, forensics, genomics, proteomics and other fields.
  • the technology provides compact units that may be deployed in the field to serve, for example, as pathogen monitoring devices for buildings, highways, cities, states, planes, airports, ships, or ports.
  • the systems can prepare and analyze samples from air, biological fluids, agricultural products, or other sources to detect target pathogens.
  • the combination of low consumable costs with automated preparation and analysis provides for a high throughput analytical system capable of screening a large number of samples simultaneously and identifying the presence or absence of a test analyte in the samples with high specificity.
  • the systems disclosed herein also can be applied to pharmacogenetics, human medical genetics, biomedical research, animal and plant typing, and human identification.
  • Additional applications of the disclosed systems include molecular diagnostics, such as detecting microorganisms, genotyping organisms, sequencing, and forensics; creating sample preparation and analysis platforms for various methodologies, such as RT-PCR, sequencing, amino acid sequence detection, protein analysis, mass spectrometry, capillary array electrophoresis, differential display, and single molecule detection.
  • molecular diagnostics such as detecting microorganisms, genotyping organisms, sequencing, and forensics
  • sample preparation and analysis platforms for various methodologies, such as RT-PCR, sequencing, amino acid sequence detection, protein analysis, mass spectrometry, capillary array electrophoresis, differential display, and single molecule detection.
  • the two stage approach to detection of a target sequence in a specimen increases substantially the specificity of detection and greatly diminishes the false positives and false negatives of the measurement. Increased specificity comes about through the multiplicative combination of specificity for each detection stage.
  • a signal is deemed a true positive based on observation of a positive signal in Stage 1 and a positive signal in Stage 2. To estimate this improvement, assume in both Stage 1 and Stage 2 the PCR assay amplifies three targets per micro-organism.
  • the invention provides specificity associated with 10 3 sensors x 10 4 measurements/sensor.
  • Stage 2 A similar specificity is observed for Stage 2 with a combined specificity of 10 9 x 10 9 or 10 18 (e.g., at least about 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 ).
  • a high specificity is important in the context of detection of biowarfare agents where a high false positive or false negative rate would make the detection system unreliable and unusable.
  • the sensitivity of the two stage system would be less than about 100 copies per micro-organism and up to at least 20 different micro-organisms per sample could be detected based on the multiplex-demultiplexed detection capability of the system. In particular, 200 copy x 100 bp/copy / (Ie "8 g / 650 g/mole * 6e23 bp/mole).
  • the two stage system design increases specificity and sensitivity without increasing system and measurement costs and combines high specificity and sensitivity in a single system.
  • the invention provides for the assay of at least about 10, 20, 50, 100, 150, 175, 200, 250, 300, 500, or even 1000 targets in a 5-10 ng DNA sample.
  • the invention provides for the analysis of at least about 3, 5, 10, 20, 30, 40 or 50 threats (e.g., pathogens) in a single sample.
  • the samples are processed in batches or are processed continuously.
  • the time to answer is quick (less than about 1 hour, 2 hours, 3, hours) and the multiplexed initial detection combined with de-multiplexed, specific detection in the second stage enables detection of multiple pathogens simultaneously and is readily scalable to detect larger numbers of micro-organisms.
  • the two stage system design intrinsically provides the high specificity and sensitivity needed for reliable and robust detection of a multitude of pathogenic organisms.
  • bacterial and viral pathogens may be detected using the system and methods of the invention.
  • Exemplary bacterial pathogens include, but are not limited to, Aerobacter, Aeromonas, Acinetobacter, Actinomyces israelii, Agrobacterium, Bacillus, Bacillus antracis, Bacteroides, Bartonella, Bordetella, Bortella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Clostridium perfringers, Clostridium tetani, Cornyebacterium, corynebacterium diphtheriae, corynebacterium sp.
  • Enterobacter Enterobacter aerogenes, Enterococcus, Erysipelothrix rhusiopathiae, Escherichia, Francisella, Fusobacterium nucleatum, Gardnerella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Klebsiella pneumoniae, Lactobacillus, Legionella, Leptospira, Listeria, Morganella, Moraxella, Mycobacterium, Neisseria, Pasteurella, Pasturella multocida, Proteus, Providencia, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus, Stentorophomonas, Streptococcus, Streptobacillus moniliformis, Treponema, Treponema pallidium, Treponema per ***, Xanthomonas, Vibrio, and Yersinia.
  • Retroviridae e.g. human immunodeficiency viruses, such as HIV-I (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses);
  • Retroviridae e.g. human immunodeficiency viruses, such as HIV-I (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates
  • Rhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g. ebola viruses
  • Paramyxoviridae e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses
  • Arena viridae hemorrhagic fever viruses
  • Reoviridae e.g.
  • reoviruses reoviruses, orbiviurses and rotaviruses
  • Birnaviridae Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g.
  • Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii.
  • Blood-borne and/or tissues parasites include Plasmodium spp., Babesia microti, Babesia diver gens, Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.
  • the invention provides systems and methods for the detection of pathogens that may be employed as biological weapons. Exemplary organisms that may be employed as biological weapons are provided in Table 1 (below). Table 1
  • Viral encephalitides Rickettsia prowazekii Viral hemorrhagic fevers b (typhus) Yersinia pest ⁇ s b Salmonella Typhimurium
  • Salmonella typhi Salmonella typhi
  • Real-Time PCR performance uniformity on the OpenArrayTM platform was detected by conducting loading of 3072 through-holes with identical primer pair/sample combination.
  • the experiment included three OpenArrayTM plates cycled in parallel, resulting in 9216 data points.
  • the data obtained from this experiment indicated highly reproducible Real-Time PCR on the OpenArrayTM platform.
  • the standard deviation calculated across over 9000 data points was 0.1 ICt for 1000 target copies/through-hole, and 0.21Ct for 100 copies/through- hole ( Figures 8 A and 8B).
  • Validated primer sequences are loaded onto OpenArrayTM plates. Standard PCR procedures, except the samples and master mix, are loaded onto OpenArrayTM plates instead of other media.
  • researchers then seal the plates in glass cases, thermal cycle in the OpenArrayTM NT Cycler. SYBR Green I fluorescence is collected and processed into cycle threshold (CT), amplicon melt temperature (Tm) and other PCR quality scores by the OpenArrayTM NT Cycler Real-Time qPCR software.
  • CT cycle threshold
  • Tm ampli
  • Assay sensitivity and specificity were determined by detecting genomic DNA from a single organism individually and in a mix of DNA from 4 or 8 organisms.
  • Real-Time PCR performed on the OATM platform allowed detection of targeted regions of Vibrio cholerae, Legionella pneumonia, Cryptosporidium parvum and Staphylococcus aureus DNA samples, isolated from pure culture as well as from a mixture of four or eight pathogens (Figure 11).
  • Real-Time PCR on the OpenArrayTM Platform is an ideal tool for highly parallel primer validation and multi-sample examination for pathogen detection.
  • Advantageously data generated on the OATM was highly efficient, reproducible and directly measured quantification via real time SYBR Green I Real-Time PCR.
  • Assay design on the OATM is quite flexible, enabling researchers to test unlimited variety of assay/sample combinations. Specificity of the assays is confirmed by dissociation curve analysis. Finally, the method is suitable for analysis of a variety of relatively low abundant microorganisms in high background DNA (i.e. environmental samples).
  • Example 3 Pri-lock probes and real-time PCR
  • PRI-lock probes for multiplex detection which provide flexibility in target specific recognition and high-throughput amplification (Szemes et al., "Diagnostic application of Padlock Probes - Multiplex Detection of Plant Pathogens using universal microarray," Nucleic Acids Research, 2005, Vol. 33, No. 8 e70).
  • PRI-lock probes are long circularizable oligonucleotides with artificially selected unique primer pairs and an universal TaqMan probe region, flanked by target complementary regions ( Figure 12). In this study, the quantification power of circularizable ligation probes was characterized, and a highthroughput, quantitative multiplex diagnostic assay was developed.
  • Example 4 Multiplex quantitative target detection Real-time quantification for multiple targets is performed in an OpenArray.

Abstract

La présente invention concerne des compositions et des procédés qui sont utiles pour la détection d’un analyte cible, tel qu’un pathogène, dans un échantillon.
PCT/US2008/014042 1996-04-02 2008-12-22 Système pour la détection d’un pathogène biologique et utilisation de celui-ci WO2009120183A2 (fr)

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CN105154325A (zh) * 2015-09-24 2015-12-16 中国科学院东北地理与农业生态研究所 真菌寄生大豆胞囊线虫检测装置
CN105154325B (zh) * 2015-09-24 2017-06-13 中国科学院东北地理与农业生态研究所 真菌寄生大豆胞囊线虫检测装置
WO2022067079A1 (fr) * 2020-09-24 2022-03-31 InnoTech Precision Medicine, Inc. Systèmes, appareil et procédés de détection de pathogènes
US11541393B2 (en) 2020-09-24 2023-01-03 InnoTech Precision Medicine, Inc. Systems, apparatus, and methods for detecting pathogens

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EP2231851A2 (fr) 2010-09-29
EP2231851A4 (fr) 2011-02-02
CN102015996A (zh) 2011-04-13

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