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WO2008127363A2 - Dispositifs de génération de polymères détectables - Google Patents

Dispositifs de génération de polymères détectables Download PDF

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Publication number
WO2008127363A2
WO2008127363A2 PCT/US2007/081286 US2007081286W WO2008127363A2 WO 2008127363 A2 WO2008127363 A2 WO 2008127363A2 US 2007081286 W US2007081286 W US 2007081286W WO 2008127363 A2 WO2008127363 A2 WO 2008127363A2
Authority
WO
WIPO (PCT)
Prior art keywords
locations
amplification product
primer system
primer
nucleotides
Prior art date
Application number
PCT/US2007/081286
Other languages
English (en)
Other versions
WO2008127363A3 (fr
WO2008127363A9 (fr
Inventor
Eric K. Engelhard
Original Assignee
Fair Isaac Corporation
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
Priority claimed from US11/548,997 external-priority patent/US7476505B2/en
Priority claimed from US11/548,980 external-priority patent/US7582433B2/en
Priority claimed from US11/548,989 external-priority patent/US7462456B2/en
Priority claimed from US11/549,008 external-priority patent/US7521189B2/en
Priority claimed from US11/548,986 external-priority patent/US20080090229A1/en
Priority claimed from US11/548,961 external-priority patent/US20080124710A1/en
Application filed by Fair Isaac Corporation filed Critical Fair Isaac Corporation
Priority to US12/444,120 priority Critical patent/US20100105025A1/en
Publication of WO2008127363A2 publication Critical patent/WO2008127363A2/fr
Publication of WO2008127363A9 publication Critical patent/WO2008127363A9/fr
Publication of WO2008127363A3 publication Critical patent/WO2008127363A3/fr

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Classifications

    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip

Definitions

  • This document relates to systems, devices, and methods involved in generating detectable polymers.
  • HT7900 Micro Fluidic CardTM is an example of a microfluidic card designed to allow a user to generate polymers.
  • the microfluidic card functions as a structured array of reaction chambers and contains input ports for inserting samples into the card.
  • the HT7900 Micro Fluidic CardTM is available from Applied Biosystems Group (Foster City, CA).
  • the FLx800TM reader is an example of an absorbance and fluorescence instrument for measuring samples in various microplate arrangements.
  • the reader can used in numerous fluorescence and absorbance applications in research and routine investigations. Its fluorescence filters are arranged in filter wheels.
  • the reader can handle 6, 48, 96, and 384 well plates and can detect wavelengths in the fluorescence spectral range.
  • Gen5TM data collection and analysis software can be used for data capture, and standard reads and data can be downloaded into Excel for further analysis. Dual optical channels can allow for measurements from above or below the plate. Light to and from the samples can be focused by a lens.
  • the FLx800TM reader is available from BioTek Instruments, Inc. (Winooski, VT).
  • This document relates to systems, devices, and methods involved in generating detectable polymers.
  • this document provides diagnostic systems, diagnostic devices, primer systems, and collections of primer systems.
  • a diagnostic system can include a diagnostic device containing a collection of primer systems.
  • This document also provides methods for making diagnostic systems, diagnostic devices, primer systems, and collections of primer systems.
  • this document provides methods for making a diagnostic device containing a collection of primer systems.
  • the systems, devices, and methods provided herein can be used to generate detectable polymers such as amplified deoxyribonucleic acid molecules.
  • the systems, devices, and methods provided herein can be used to detect influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses within samples.
  • Detecting viruses e.g., influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses
  • Detecting viruses can help clinicians provide important prognostic information to patients and can help epidemiologists select appropriate viruses for vaccine development.
  • the description provided herein is based, in part, on the discovery of effective primer systems for generating detectable polymers.
  • a diagnostic device can contain primer systems effective to detect viruses (e.g., influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses) within samples.
  • viruses e.g., influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses
  • viruses e.g., influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses
  • the description provided herein also is based, in part, on the discovery of primer systems having the ability to not only amplify particular nucleic acid sequences from different viruses (e.g., influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses), but also to not amplify nucleic acid sequences from non-influenza A virus, non-adenovirus, non-calicivirus, non-coronavirus, non-respiratory syncytial virus, or non-SARS coronavirus sources such as a human's genome.
  • viruses e.g., influenza A viruses, adenoviruses, caliciviruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses
  • a single diagnostic card having multiple separate microfluidic chambers, each of which contains a different primer system provided herein, can be used in a single amplification reaction to detect the presence or absence of multiple different influenza A viruses.
  • Having the ability to test for the presence or absence of multiple different influenza A viruses using a single diagnostic card with the primer systems provided herein and a single amplification reaction can allow clinicians to detect different influenza A viruses within samples (e.g., a sample collected from a human) accurately and rapidly in a cost effective manner.
  • one aspect of this document features a device comprising, or consisting essentially of, a housing having a plurality of locations, wherein each of the locations contains an influenza virus primer system, wherein the primers of each influenza virus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device comprises at least one influenza virus primer system capable of producing an amplification product diagnostic for an Hl target, at least one influenza virus primer system capable of producing an amplification product diagnostic for an H2 target, at least one influenza virus primer system capable of producing an amplification product diagnostic for an H3 target, at least one influenza virus primer system capable of producing an amplification product diagnostic for an H5 target, at least one influenza virus primer system capable of producing an amplification product diagnostic for an Nl target, and at least one influenza virus primer system capable of producing an amplification product diagnostic for an N2 target, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber. Each of the locations can be a well. Two or more of the locations can comprise two or more influenza virus primer systems. Each influenza virus primer system of the two or more influenza virus primer systems of a location can be capable of producing an amplification product diagnostic for a different target of influenza A viruses.
  • the primers of each primer system can be between 23 and 27 nucleotides in length.
  • the primers of each influenza virus primer system can have a theoretical melting temperature between 59°C and 61 0 C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • Each of the locations can comprise an intercalating dye, and each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • this document features a diagnostic device for detecting an influenza virus within a sample.
  • the device comprises, or consists essentially of, a housing having a plurality of locations, wherein each of the locations contains an influenza virus primer system, wherein the primers of each influenza virus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for each of at least six different targets of influenza A viruses, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber.
  • Each of the locations can be a well. Two or more of the locations can comprise two or more influenza virus primer systems.
  • Each influenza virus primer system of the two or more influenza virus primer systems of a location can be capable of producing an amplification product diagnostic for a different target of influenza A viruses.
  • the primers of each influenza virus primer system can be between 23 and 27 nucleotides in length.
  • the primers of each influenza virus primer system can have a theoretical melting temperature between 59°C and 61 0 C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • the at least six different targets of influenza A viruses can be Hl, H2, H3, H5, Nl, and N2 targets.
  • Each of the locations can comprise an intercalating dye, and each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • the method comprises, or consists essentially of, (a) performing a nucleic acid amplification reaction using the sample as a source of template and a diagnostic device, wherein the device comprises, or consists essentially of, a housing having a plurality of locations, wherein each of the locations contains an influenza virus primer system, wherein the primers of each influenza virus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for at least six different targets of influenza A viruses, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length, and (b) determining which locations of the device contain an influenza virus primer system that resulted in the formation of amplification product, thereby detecting an influenza A virus.
  • the sample can be a sample obtained from a human.
  • the nucleic acid amplification reaction can comprise at least 10 cycles.
  • the nucleic acid amplification reaction can comprise at least 20 cycles.
  • the nucleic acid amplification reaction can comprise a denaturing step at about 94°C or about 95°C.
  • the nucleic acid amplification reaction can comprise an annealing step at about 60 0 C.
  • the nucleic acid amplification reaction can comprise an extension step at about 72°C.
  • the sample can be a mucus sample.
  • the sample can be a sample obtained from the human using a swab.
  • the sample can be a sample processed to obtain viral nucleic acid.
  • the amplification reaction can be performed in a thermal cycler device configured to receive the diagnostic device.
  • the determining step (b) can be performed in using a dye reader device configured to receive the diagnostic device.
  • Each of the locations can comprise an intercalating dye, wherein each amplification product, when produced, is labeled with the intercalating dye, and wherein determining which locations of the device contain a primer system that resulted in the formation of amplification product is based on a signal from the dye.
  • the amplification reaction and the determining step (b) can be performed in a machine configured to receive the diagnostic device, the machine comprising a thermal cycler device and a dye reader device.
  • the machine can be capable of providing output indicating the presence of the influenza A virus.
  • the machine can be capable of providing output indicating the influenza virus primer systems that detected the presence of the influenza A virus.
  • the output can be paper printout or a computer readable file.
  • this document features a device comprising, or consisting essentially of, a housing having a plurality of locations, wherein each of the locations contains an adenovirus primer system, wherein the primers of each adenovirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device comprises at least one adenovirus primer system capable of producing an amplification product diagnostic for an adenovirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber.
  • Each of the locations can be a well.
  • the primers of each adenovirus primer system can be between 23 and 27 nucleotides in length.
  • the primers of each adenovirus primer system can have a theoretical melting temperature between 59°C and 61°C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • Each of the locations can comprise an intercalating dye, and wherein each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • this document features method for detecting an adenovirus within a sample.
  • the method comprises, or consists essentially of, (a) performing a nucleic acid amplification reaction using the sample as a source of template and a diagnostic device, wherein the device comprises a housing having a plurality of locations, wherein each of the locations contains an adenovirus primer system, wherein the primers of each adenovirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for an adenovirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length, and (b) determining which locations of the device contain an adenovirus primer system that resulted in the formation of amplification product, thereby detecting an adenovirus.
  • the sample can be a sample obtained from a human.
  • the nucleic acid amplification reaction can comprise at least 10 cycles.
  • the nucleic acid amplification reaction can comprise at least 20 cycles.
  • the nucleic acid amplification reaction can comprise a denaturing step at about 94°C or about 95°C.
  • the nucleic acid amplification reaction can comprise an annealing step at about 60 0 C.
  • the nucleic acid amplification reaction can comprise an extension step at about 72°C.
  • the sample can be a mucus sample.
  • the sample can be a sample obtained from the human using a swab.
  • the sample can be a sample processed to obtain viral nucleic acid.
  • Each of the locations can comprise an intercalating dye, wherein each amplification product, when produced, is labeled with the intercalating dye, and wherein determining which locations of the device contain an adenovirus primer system that resulted in the formation of amplification product is based on a signal from the dye.
  • the amplification reaction can be performed in a thermal cycler device configured to receive the diagnostic device.
  • the determining step (b) can be performed in using a dye reader device configured to receive the diagnostic device.
  • the amplification reaction and the determining step (b) can be performed in a machine configured to receive the diagnostic device, the machine comprising a thermal cycler device and a dye reader device. The machine can be capable of providing output indicating the presence of the adenovirus.
  • the machine can be capable of providing output indicating the adenovirus primer system that detected the presence of the adenovirus.
  • the output can be a paper printout or a computer readable file.
  • this document features a device comprising, or consisting essentially of, a housing having a plurality of locations, wherein each of the locations contains a calicivirus primer system, wherein the primers of each calicivirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device comprises at least one calicivirus primer system capable of producing an amplification product diagnostic for an calicivirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber. Each of the locations can be a well.
  • the primers of each calicivirus primer system can be between 23 and 27 nucleotides in length.
  • the primers of each calicivirus primer system can have a theoretical melting temperature between 59°C and 61 0 C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • Each of the locations can comprise an intercalating dye, and wherein each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • this document features method for detecting a calicivirus within a sample.
  • the method comprises, or consists essentially of, (a) performing a nucleic acid amplification reaction using the sample as a source of template and a diagnostic device, wherein the device comprises a housing having a plurality of locations, wherein each of the locations contains a calicivirus primer system, wherein the primers of each calicivirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for a calicivirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length, and (b) determining which locations of the device contain a calicivirus primer system that resulted in the formation of amplification product, thereby detecting a calicivirus.
  • the sample can be a sample obtained from a human.
  • the nucleic acid amplification reaction can comprise at least 10 cycles.
  • the nucleic acid amplification reaction can comprise at least 20 cycles.
  • the nucleic acid amplification reaction can comprise a denaturing step at about 94°C or about 95°C.
  • the nucleic acid amplification reaction can comprise an annealing step at about 60 0 C.
  • the nucleic acid amplification reaction can comprise an extension step at about 72°C.
  • the sample can be a mucus sample.
  • the sample can be a sample obtained from the human using a swab.
  • the sample can be a sample processed to obtain viral nucleic acid.
  • Each of the locations can comprise an intercalating dye, wherein each amplification product, when produced, is labeled with the intercalating dye, and wherein determining which locations of the device contain a primer system that resulted in the formation of amplification product is based on a signal from the dye.
  • the amplification reaction can be performed in a thermal cycler device configured to receive the diagnostic device.
  • the determining step (b) can be performed in using a dye reader device configured to receive the diagnostic device.
  • the amplification reaction and the determining step (b) can be performed in a machine configured to receive the diagnostic device, the machine comprising a thermal cycler device and a dye reader device.
  • the machine can be capable of providing output indicating the presence of the calicivirus.
  • the machine can be capable of providing output indicating the calicivirus primer system that detected the presence of the calicivirus.
  • the output can be a paper printout or a computer readable file.
  • this document features a device comprising, or consisting essentially of, a housing having a plurality of locations, wherein each of the locations contains a coronavirus primer system, wherein the primers of each coronavirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device comprises at least one primer system capable of producing an amplification product diagnostic for a coronavirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber.
  • Each of the locations can be a well.
  • the primers of each coronavirus primer system can be between 23 and 27 nucleotides in length.
  • the primers of each coronavirus primer system can have a theoretical melting temperature between 59°C and 61 0 C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • Each of the locations can comprise an intercalating dye, and wherein each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • this document features method for detecting a coronavirus within a sample.
  • the method comprises, or consists essentially of, (a) performing a nucleic acid amplification reaction using the sample as a source of template and a diagnostic device, wherein the device comprises a housing having a plurality of locations, wherein each of the locations contains a coronavirus primer system, wherein the primers of each coronavirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for a coronavirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length, and (b) determining which locations of the device contain a primer system that resulted in the formation of amplification product, thereby detecting a coronavirus.
  • the sample can be a sample obtained from a human.
  • the nucleic acid amplification reaction can comprise at least 10 cycles.
  • the nucleic acid amplification reaction can comprise at least 20 cycles.
  • the nucleic acid amplification reaction can comprise a denaturing step at about 94°C or about 95°C.
  • the nucleic acid amplification reaction can comprise an annealing step at about 60 0 C.
  • the nucleic acid amplification reaction can comprise an extension step at about 72°C.
  • the sample can be a mucus sample.
  • the sample can be a sample obtained from the human using a swab.
  • the sample can be a sample processed to obtain viral nucleic acid.
  • Each of the locations can comprise an intercalating dye, wherein each amplification product, when produced, is labeled with the intercalating dye, and wherein determining which locations of the device contain a primer system that resulted in the formation of amplification product is based on a signal from the dye.
  • the amplification reaction can be performed in a thermal cycler device configured to receive the diagnostic device.
  • the determining step (b) can be performed in using a dye reader device configured to receive the diagnostic device.
  • the amplification reaction and the determining step (b) can be performed in a machine configured to receive the diagnostic device, the machine comprising a thermal cycler device and a dye reader device. The machine can be capable of providing output indicating the presence of the coronavirus.
  • the machine can be capable of providing output indicating the coronavirus primer system that detected the presence of the coronavirus.
  • the output can be a paper printout or a computer readable file.
  • this document features a device comprising, or consisting essentially of, a housing having a plurality of locations, wherein each of the locations contains a respiratory syncytial virus primer system, wherein the primers of each respiratory syncytial virus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device comprises at least one respiratory syncytial virus primer system capable of producing an amplification product diagnostic for a respiratory syncytial virus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber. Each of the locations can be a well.
  • the primers of each respiratory syncytial virus primer system can be between 23 and 27 nucleotides in length.
  • the primers of each respiratory syncytial virus primer system can have a theoretical melting temperature between 59°C and 61 0 C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • Each of the locations can comprise an intercalating dye, and wherein each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • this document features method for detecting a respiratory syncytial virus within a sample.
  • the method comprises, or consists essentially of, (a) performing a nucleic acid amplification reaction using the sample as a source of template and a diagnostic device, wherein the device comprises a housing having a plurality of locations, wherein each of the locations contains a respiratory syncytial virus primer system, wherein the primers of each respiratory syncytial virus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for a respiratory syncytial virus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length, and (b) determining which locations of the device contain a primer system that resulted in the formation of amplification product, thereby detecting a respiratory syncytial virus.
  • the sample can be a sample obtained from a human.
  • the nucleic acid amplification reaction can comprise at least 10 cycles.
  • the nucleic acid amplification reaction can comprise at least 20 cycles.
  • the nucleic acid amplification reaction can comprise a denaturing step at about 94°C or about 95°C.
  • the nucleic acid amplification reaction can comprise an annealing step at about 60 0 C.
  • the nucleic acid amplification reaction can comprise an extension step at about 72°C.
  • the sample can be a mucus sample.
  • the sample can be a sample obtained from the human using a swab.
  • the sample can be a sample processed to obtain viral nucleic acid.
  • Each of the locations can comprise an intercalating dye, wherein each amplification product, when produced, is labeled with the intercalating dye, and wherein determining which locations of the device contain a primer system that resulted in the formation of amplification product is based on a signal from the dye.
  • the amplification reaction can be performed in a thermal cycler device configured to receive the diagnostic device.
  • the determining step (b) can be performed in using a dye reader device configured to receive the diagnostic device.
  • the amplification reaction and the determining step (b) can be performed in a machine configured to receive the diagnostic device, the machine comprising a thermal cycler device and a dye reader device.
  • the machine can be capable of providing output indicating the presence of the respiratory syncytial virus.
  • the machine can be capable of providing output indicating the respiratory syncytial virus primer system that detected the presence of the respiratory syncytial virus.
  • the output can be a paper printout or a computer readable file.
  • this document features a device comprising, or consisting essentially of, a housing having a plurality of locations, wherein each of the locations contains a SARS coronavirus primer system, wherein the primers of each SARS coronavirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device comprises at least one SARS coronavirus primer system capable of producing an amplification product diagnostic for a SARS coronavirus, and wherein each amplification product, when produced, is between 100 and 400 nucleotides in length.
  • Each of the locations can be a chamber.
  • Each of the locations can be a well.
  • the primers of each SARS coronavirus primer system can be between 23 and 27 nucleotides in length.
  • the primers of each SARS coronavirus primer system can have a theoretical melting temperature between 59°C and 61°C.
  • the housing can comprise additional locations, wherein each of the additional locations contains a primer pair. At least one of the additional locations can comprise a primer pair capable of producing an amplification product from human nucleic acid.
  • Each of the locations can comprise an intercalating dye, and wherein each amplification product, when produced, can be labeled with the intercalating dye.
  • the intercalating dye can be a green fluorescent dye.
  • the intercalating dye can be SYBR Green, LC Green, or SYTO9.
  • Each amplification product, when produced, can be between 100 and 300 nucleotides in length.
  • this document features method for detecting a SARS coronavirus within a sample.
  • the method comprises, or consists essentially of, (a) performing a nucleic acid amplification reaction using the sample as a source of template and a diagnostic device, wherein the device comprises a housing having a plurality of locations, wherein each of the locations contains a SARS coronavirus primer system, wherein the primers of each SARS coronavirus primer system are between 18 and 28 nucleotides in length and have a theoretical melting temperature between 58°C and 62°C, wherein the device is capable of producing an amplification product diagnostic for a
  • each amplification product, when produced, is between 100 and 400 nucleotides in length, and (b) determining which locations of the device contain a primer system that resulted in the formation of amplification product, thereby detecting a SARS coronavirus.
  • the sample can be a sample obtained from a human.
  • the nucleic acid amplification reaction can comprise at least 10 cycles.
  • the nucleic acid amplification reaction can comprise at least 20 cycles.
  • the nucleic acid amplification reaction can comprise a denaturing step at about 94°C or about 95°C.
  • the nucleic acid amplification reaction can comprise an annealing step at about 60 0 C.
  • the nucleic acid amplification reaction can comprise an extension step at about 72°C.
  • the sample can be a mucus sample.
  • the sample can be a sample obtained from the human using a swab.
  • the sample can be a sample processed to obtain viral nucleic acid.
  • Each of the locations can comprise an intercalating dye, wherein each amplification product, when produced, is labeled with the intercalating dye, and wherein determining which locations of the device contain a primer system that resulted in the formation of amplification product is based on a signal from the dye.
  • the amplification reaction can be performed in a thermal cycler device configured to receive the diagnostic device.
  • the determining step (b) can be performed in using a dye reader device configured to receive the diagnostic device.
  • the amplification reaction and the determining step (b) can be performed in a machine configured to receive the diagnostic device, the machine comprising a thermal cycler device and a dye reader device.
  • the machine can be capable of providing output indicating the presence of the SARS coronavirus.
  • the machine can be capable of providing output indicating the SARS coronavirus primer system that detected the presence of the SARS coronavirus.
  • the output can be a paper printout or a computer readable file.
  • FIG. 1 is a top view of a microfluidic card.
  • This document provides systems, devices, and methods involved in generating detectable polymers.
  • this document provides diagnostic systems, diagnostic devices, primer systems, and collections of primer systems.
  • a diagnostic system can include a diagnostic device containing primer systems.
  • a diagnostic device can include a housing having a plurality of locations.
  • the housing can be any shape and size and can be made from any type of material including, without limitation, plastic, glass, silicone, or metal.
  • a housing provided herein can be rectangular, square, circular, or oval in shape, and can have a length, width, or diameter between five cm and 50 cm (e.g., between ten cm and 40 cm, between ten cm and 30 cm, or between ten cm and 25 cm).
  • the depth or height of a housing provided herein can be between 0.2 cm and 2 cm (e.g., between 0.2 and 1 cm, between 0.3 and 1 cm, or between 0.5 and 1 cm).
  • Each location of a housing can be configured to allow an amplification reaction to occur without primer system contamination from other locations.
  • the locations of a housing provided herein can be any shape or size.
  • the locations of a housing provided herein can be in the configuration of a well or chamber with, for example, the ability to hold a volume between 1 ⁇ L and 100 ⁇ L (e.g., between 1 ⁇ L and 20 ⁇ L, between 1 ⁇ L and 10 ⁇ L, between 1 ⁇ L and 5 ⁇ L, between 10 ⁇ L and 50 ⁇ L, or between 15 ⁇ L and 25 ⁇ L).
  • a volume can be 1.5 ⁇ L, 10 ⁇ L, 20 ⁇ L, or 30 ⁇ L.
  • a housing can be a 96-well plate with each location being a well of the 96-well plate.
  • a diagnostic device can be in the form of a micro fluidic card.
  • Such a card can have a series of locations and channels. The channels can provide fluid communication between a sample inlet port and one or more locations.
  • a housing can be a mirco fluidic card having one or more sample inlet ports in fluid communication with one or more locations via one or more channels.
  • such a housing can include one or more outlet ports for providing an outlet for added solutions or for providing an outlet for air so that fluid can flow through the channels.
  • a diagnostic device provided herein can be in the form of a micro fluidic card with eight sample inlet ports each connected through channels (e.g., microcapillaries) to 48 locations (e.g., reaction chambers).
  • channels e.g., microcapillaries
  • locations e.g., reaction chambers.
  • micro fluidic card 100 can have housing 102 defining a plurality of locations 106. While 280 separate locations are shown in this example, a housing provided herein can define any number of locations (e.g., 10, 25, 48, 96, 384, 1536, or more locations). Each location 106 can be in fluid communication with a sample inlet port 104 and an outlet port 108 via channel 110. Any number of channels can be defined by housing 102. For example, a housing provided herein can define one continuous, interconnected channel or can contain multiple separate channels.
  • a diagnostic device provided herein can contain a collection of primer systems and primer pairs.
  • each primer system or primer pair of a collection can be located at a different location defined by a housing so as to isolate each primer system or primer pair from the other primer systems or primer pairs of the collection.
  • each primer system or primer pair of a collection can be housed within a separate location (e.g., a separate well of a plastic microtiter plate or a separate chamber of a micro fluidic card).
  • each primer system or primer pair of a collection or a subset of primer systems or primer pairs of a collection can be housed together.
  • one primer system provided herein and one primer pair of a collection of 50 primer systems and primer pairs can be housed within a single well of a plastic microtiter plate with the remaining 48 primer systems and primer pairs being housed within separate wells.
  • a system or diagnostic device can contain at least six different primer systems set forth in Table 1 (e.g., at least seven, at least eight, at least nine, at least ten, at least eleven, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 different primer systems set forth in Table 1).
  • a diagnostic device can contain primer systems not listed in Table 1.
  • a diagnostic device can contain a primer system similar to primer system number 1 with the exception that each nucleic acid primer is two nucleotides shorter than those of primer system number 1.
  • a diagnostic device can contain a primer pair designed to amplify host nucleic acid (e.g., human genomic nucleic acid or mRNA).
  • each primer of an influenza virus primer system can be from 15 to 45 nucleotides the length.
  • each primer of an influenza virus primer system provided herein can range from 20 to 40 nucleotides (e.g., from 20 to 35 nucleotides, from 20 to 30 nucleotides, or from 21 to 28 nucleotides).
  • influenza virus primer systems provided herein can be selected such that the length of amplified target nucleic acid, if present within an amplification reaction, would be between 100 and 400 nucleotides (e.g., between 150 and 350 nucleotides, between 175 and 325 nucleotides, or between 200 and 300 nucleotides).
  • the theoretical melting temperature of each primer of an influenza virus primer system provided herein can be between 58°C and 62°C (e.g., between 59°C and 6PC).
  • influenza virus primer system can be used to amplify nucleic acid present in an influenza virus (e.g., influenza A virus).
  • influenza virus primer systems provided herein can share unifying advantageous features.
  • each influenza virus primer system provided herein can amplify nucleic acid from influenza A viruses.
  • influenza virus primer systems provided herein can be selected such that the length of amplified viral nucleic acid would be between 100 and 400 nucleotides.
  • the theoretical melting temperature of the influenza virus primer systems provided herein can be uniformly between 58°C and 62°C, and the length of each primer of the influenza virus primer systems provided herein can range from 15 to 50 nucleotides (e.g., from 21 to 28 nucleotides).
  • the first primer system listed in Table 1 for each of the different targets can be used to make a collection of at least six different primer systems and can be used effectively to detect a large group of different influenza A viruses.
  • primer system number 1 can have the ability to detect Hl nucleic acid sequences associated with 457 different GenBank gi numbers.
  • Primer system number 4 can have the ability to detect H2 nucleic acid sequences associated with 62 different GenBank gi numbers.
  • Primer system number 7 can have the ability to detect H3 nucleic acid sequences associated with 1842 different GenBank gi numbers.
  • Primer system number 10 can have the ability to detect H5 nucleic acid sequences associated with 497 different GenBank gi numbers.
  • Primer system number 13 can have the ability to detect Nl nucleic acid sequences associated with 430 different GenBank gi numbers.
  • Primer system number 16 can have the ability to detect N2 nucleic acid sequences associated with 1050 different GenBank gi numbers.
  • a system or diagnostic device provided herein can contain at least one primer system set forth in Table 3 (e.g., at least two primer systems set forth in Table 3).
  • a diagnostic device can contain primer systems not listed in Table 3.
  • a diagnostic device can contain a primer system similar to primer system number 19 with the exception that each nucleic acid primer is two nucleotides shorter than those of primer system number 19.
  • a diagnostic device can contain a primer pair designed to amplify host nucleic acid (e.g., human genomic nucleic acid or mRNA).
  • adenovirus primer system refers to a combination of two nucleic acid primers having the ability to amplify nucleic acid provided that the sequence of each nucleic acid primer is from 15 to 50 nucleotides in length and is such that it aligns without a mismatch to a sequence, or its complement, set forth in a GenBank gi number listed in Table 4.
  • each primer of an adenovirus primer system provided herein can be from 15 to 45 nucleotides the length.
  • each primer of an adenovirus primer system provided herein can range from 20 to 40 nucleotides (e.g., from 20 to 35 nucleotides, from 20 to 30 nucleotides, or from 21 to 28 nucleotides).
  • the adenovirus primer systems provided herein can be selected such that the length of amplified target nucleic acid, if present within an amplification reaction, would be between 100 and 400 nucleotides (e.g., between 150 and 350 nucleotides, between 175 and 325 nucleotides, or between 200 and 300 nucleotides).
  • the theoretical melting temperature of each primer of an adenovirus primer system provided herein can be between 58°C and 62°C (e.g., between 59°C and 61°C).
  • Each adenovirus primer system provided herein can be used to amplify nucleic acid present in an adenovirus.
  • each adenovirus primer system provided herein can amplify nucleic acid from adenoviruses.
  • adenovirus primer systems provided herein can be selected such that the length of amplified viral nucleic acid would be between 100 and 400 nucleotides.
  • the theoretical melting temperature of the adenovirus primer systems provided herein can be uniformly between 58°C and 62°C, and the length of each primer of the adenovirus primer systems provided herein can range from 15 to 50 nucleotides (e.g., from 21 to 28 nucleotides).
  • adenovirus primer systems listed in Table 3 can be used effectively to detect a large group of different adenoviruses.
  • adenovirus primer system number 19 can have the ability to detect adenovirus nucleic acid sequences associated with 89 different GenBank gi numbers.
  • a system or diagnostic device provided herein can contain at least one primer system set forth in Table 5 (e.g., at least two primer systems set forth in Table 5).
  • a diagnostic device can contain primer systems not listed in Table 5.
  • a diagnostic device can contain a primer system similar to primer system number 22 with the exception that each nucleic acid primer is two nucleotides shorter than those of primer system number 22.
  • a diagnostic device can contain a primer pair designed to amplify host nucleic acid (e.g., human genomic nucleic acid or mRNA).
  • calicivirus primer system refers to a combination of two nucleic acid primers having the ability to amplify nucleic acid provided that the sequence of each nucleic acid primer is from 15 to 50 nucleotides in length and is such that it aligns without a mismatch to a sequence, or its complement, set forth in a GenBank gi number listed in Table 6.
  • each primer of a calicivirus primer system provided herein can be from 15 to 45 nucleotides the length.
  • each calicivirus primer of a primer system provided herein can range from 20 to 40 nucleotides (e.g., from 20 to 35 nucleotides, from 20 to 30 nucleotides, or from 21 to 28 nucleotides).
  • the calicivirus primer systems provided herein can be selected such that the length of amplified target nucleic acid, if present within an amplification reaction, would be between 100 and 400 nucleotides (e.g., between 150 and 350 nucleotides, between 175 and 325 nucleotides, or between 200 and 300 nucleotides).
  • the theoretical melting temperature of each primer of a calicivirus primer system provided herein can be between 58°C and 62°C (e.g., between 59°C and 61°C).
  • Each calicivirus primer system provided herein can be used to amplify nucleic acid present in a calicivirus.
  • Table 6 Representative gi numbers for each calicivirus primer system.
  • each calicivirus primer system provided herein can amplify nucleic acid from caliciviruses.
  • primer systems provided herein can be selected such that the length of amplified viral nucleic acid would be between 100 and 400 nucleotides.
  • the theoretical melting temperature of the calicivirus primer systems provided herein can be uniformly between 58°C and 62°C, and the length of each primer of the calicivirus primer systems provided herein can range from 15 to 50 nucleotides (e.g., from 21 to 28 nucleotides).
  • the primer systems listed in Table 5 can be used effectively to detect a large group of different caliciviruses.
  • primer system number 22 can have the ability to detect calicivirus nucleic acid sequences associated with 36 different GenBank gi numbers.
  • a system or diagnostic device provided herein can contain at least one primer system set forth in Table 7 (e.g., at least two primer systems set forth in Table 7).
  • a diagnostic device can contain primer systems not listed in Table 7.
  • a diagnostic device can contain a primer system similar to primer system number 25 with the exception that each nucleic acid primer is two nucleotides shorter than those of primer system number 25.
  • a diagnostic device can contain a primer pair designed to amplify host nucleic acid (e.g., human genomic nucleic acid or mRNA).
  • coronavirus primer system refers to a combination of two nucleic acid primers having the ability to amplify nucleic acid provided that the sequence of each nucleic acid primer is from 15 to 50 nucleotides in length and is such that it aligns without a mismatch to a sequence, or its complement, set forth in a GenBank gi number listed in Table 8.
  • each primer of a coronavirus primer system provided herein can be from 15 to 45 nucleotides the length.
  • each primer of a coronavirus primer system provided herein can range from 20 to 40 nucleotides (e.g., from 20 to 35 nucleotides, from 20 to 30 nucleotides, or from 21 to 28 nucleotides).
  • the coronavirus primer systems provided herein can be selected such that the length of amplified target nucleic acid, if present within an amplification reaction, would be between 100 and 400 nucleotides (e.g., between 150 and 350 nucleotides, between 175 and 325 nucleotides, or between 200 and 300 nucleotides).
  • the theoretical melting temperature of each primer of a coronavirus primer system provided herein can be between 58°C and 62°C (e.g., between 59°C and 61°C).
  • Each coronavirus primer system provided herein can be used to amplify nucleic acid present in a coronavirus.
  • each coronavirus primer system provided herein can amplify nucleic acid from coronaviruses.
  • coronavirus primer systems provided herein can be selected such that the length of amplified viral nucleic acid would be between 100 and 400 nucleotides.
  • the theoretical melting temperature of the coronavirus primer systems provided herein can be uniformly between 58°C and 62°C, and the length of each primer of the coronavirus primer systems provided herein can range from 15 to 50 nucleotides (e.g., from 21 to 28 nucleotides).
  • primer system number 25 can have the ability to detect coronavirus nucleic acid sequences associated with 64 different GenBank gi numbers.
  • a system or diagnostic device provided herein can contain at least one primer system set forth in Table 9 (e.g., at least two primer systems set forth in Table 9).
  • a diagnostic device can contain primer systems not listed in Table 9.
  • a diagnostic device can contain a primer system similar to primer system number 28 with the exception that each nucleic acid primer is two nucleotides shorter than those of primer system number 28.
  • a diagnostic device can contain a primer pair designed to amplify host nucleic acid (e.g., human genomic nucleic acid or mRNA). Table 9. Optimal respiratory syncytial virus primer systems for respiratory syncytial viruses.
  • respiratory syncytial virus primer system refers to a combination of two nucleic acid primers having the ability to amplify nucleic acid provided that the sequence of each nucleic acid primer is from 15 to 50 nucleotides in length and is such that it aligns without a mismatch to a sequence, or its complement, set forth in a GenBank gi number listed in Table 10.
  • each primer of a respiratory syncytial virus primer system provided herein can be from 15 to 45 nucleotides the length.
  • each primer of a respiratory syncytial virus primer system can range from 20 to 40 nucleotides (e.g., from 20 to 35 nucleotides, from 20 to 30 nucleotides, or from 21 to 28 nucleotides).
  • the primer systems provided herein can be selected such that the length of amplified target nucleic acid, if present within an amplification reaction, would be between 100 and 400 nucleotides (e.g., between 150 and 350 nucleotides, between 175 and 325 nucleotides, or between 200 and 300 nucleotides).
  • the theoretical melting temperature of each primer of a respiratory syncytial virus primer system provided herein can be between 58°C and 62°C (e.g., between 59°C and 61°C).
  • Each respiratory syncytial virus primer system provided herein can be used to amplify nucleic acid present in a respiratory syncytial virus.
  • each respiratory syncytial virus primer system provided herein can amplify nucleic acid from respiratory syncytial viruses.
  • respiratory syncytial virus primer systems provided herein can be selected such that the length of amplified viral nucleic acid would be between 100 and 400 nucleotides.
  • the theoretical melting temperature of the respiratory syncytial virus primer systems provided herein can be uniformly between 58°C and 62°C, and the length of each primer of the respiratory syncytial virus primer systems provided herein can range from 15 to 50 nucleotides (e.g., from 21 to 28 nucleotides).
  • primer system number 28 can have the ability to detect respiratory syncytial virus nucleic acid sequences associated with 330 different GenBank gi numbers.
  • a system or diagnostic device provided herein can contain at least one primer system set forth in Table 11 (e.g., at least two primer systems set forth in Table 11).
  • a diagnostic device can contain primer systems not listed in Table 11.
  • a diagnostic device can contain a primer system similar to primer system number 31 with the exception that each nucleic acid primer is two nucleotides shorter than those of primer system number 31.
  • a diagnostic device can contain a primer pair designed to amplify host nucleic acid (e.g., human genomic nucleic acid or mRNA). Table 11. O timal SARS coronavirus rimer s stems for SARS coronaviruses.
  • SARS coronavirus primer system refers to a combination of two nucleic acid primers having the ability to amplify nucleic acid provided that the sequence of each nucleic acid primer is from 15 to 50 nucleotides in length and is such that it aligns without a mismatch to a sequence, or its complement, set forth in a GenBank gi number listed in Table 12.
  • each primer of a SARS coronavirus primer system provided herein can be from 15 to 45 nucleotides the length.
  • each primer of a SARS coronavirus primer system provided herein can range from 20 to 40 nucleotides (e.g., from 20 to 35 nucleotides, from 20 to 30 nucleotides, or from 21 to 28 nucleotides).
  • the SARS coronavirus primer systems provided herein can be selected such that the length of amplified target nucleic acid, if present within an amplification reaction, would be between 100 and 400 nucleotides (e.g., between 150 and 350 nucleotides, between 175 and 325 nucleotides, or between 200 and 300 nucleotides).
  • the theoretical melting temperature of each primer of a SARS coronavirus primer system provided herein can be between 58°C and 62°C (e.g., between 59°C and 61 0 C).
  • Each SARS coronavirus primer system provided herein can be used to amplify nucleic acid present in a SARS coronavirus.
  • each SARS coronavirus primer system provided herein can amplify nucleic acid from SARS coronaviruses.
  • SARS coronavirus primer systems provided herein can be selected such that the length of amplified viral nucleic acid would be between 100 and 400 nucleotides.
  • the theoretical melting temperature of the SARS coronavirus primer systems provided herein can be uniformly between 58°C and 62°C, and the length of each primer of the SARS coronavirus primer systems provided herein can range from 15 to 50 nucleotides (e.g., from 21 to 28 nucleotides).
  • primer system number 31 can have the ability to detect SARS coronavirus nucleic acid sequences associated with 244 different GenBank gi numbers.
  • any method can be used to make the primers of a primer system provided herein.
  • chemical synthesis techniques such as those described elsewhere (Beaucage and Caruthers, Tetrahedron Lett., 22:1859-62 (1981)) can be used.
  • nucleic acid primers can be obtained from commercial vendors such as MWG Biotech, Invitrogen, and Operon.
  • a diagnostic device provided herein can be made as follows.
  • a 384-well master plate containing 125 ⁇ L of one or more primer systems in dioinized water at a working concentration of 100 nmole/1 ⁇ L of each primer can be constructed.
  • the master plate can be used as a template source, and 1 ⁇ L of each master plate well can be transferred to corresponding wells on a 384-well micro fluidic card.
  • Spotted reagents can be allowed to dry at room temperature before the final plastic laminate layer of the microfluidic card is attached.
  • the primer systems provided herein can be used separately or in combinations. Such combinations can contain 2, 3, 4, 5, 10, 15, or more different primer systems listed in Table 1, 3, 5, 7, 9, or 11. When making a combination, any two or more of the provided primer systems can be arranged into any combination.
  • the first primer system listed in Table 1 for each of the targets can be used to make a collection of six different primer systems.
  • Other combinations include, without limitation, a combination of six different primer systems containing the second primer system listed in Table 1 for each of the targets; a combination of six different primer systems containing the third primer system listed in Table 1 for each of the targets; and a combination of 18 different primer systems containing the first, second, and third primer system listed in Table 1 for each of the targets.
  • the diagnostic devices and primer systems provided herein can be used to detect viruses (e.g., influenza A viruses, adenoviruses, calici viruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses) present within samples.
  • viruses e.g., influenza A viruses, adenoviruses, calici viruses, coronaviruses, respiratory syncytial viruses, or SARS coronaviruses
  • a sample can be obtained from a human (or other animal such as a bird) and used in an amplification reaction to determine whether or not a viral nucleic acid (e.g., an influenza A virus' nucleic acid, an adenovirus' nucleic acid, a calicivirus' nucleic acid, a coronavirus' nucleic acid, a respiratory syncytial virus' nucleic acid, or a SARS coronavirus' nucleic acid) is present in the sample.
  • a viral nucleic acid e.g., an influenza A virus' nucleic acid, an adenovirus' nucleic acid, a calicivirus' nucleic acid, a coronavirus' nucleic acid, a respiratory syncytial virus' nucleic acid, or a SARS coronavirus' nucleic acid
  • sample can be used including, without limitation, a biopsy (e.g., punch biopsy, aspiration biopsy, excision biopsy, needle biopsy, or shave biopsy), a tissue section, lymph fluid, mucus, blood, serum, and saliva samples.
  • a sample can be obtained from a human or any other animal (e.g., birds, pigs, and horses) suspected to contain a virus (e.g., an influenza A virus, an adenovirus, a calicivirus, a coronavirus, a respiratory syncytial virus, or a SARS coronavirus).
  • a virus e.g., an influenza A virus, an adenovirus, a calicivirus, a coronavirus, a respiratory syncytial virus, or a SARS coronavirus.
  • a sample can be obtained from a mammal (e.g., a human) using a swab (e.g., an OmniSwab; Whatman).
  • a swab e.g., an OmniSwab; Whatman.
  • the presence of an amplification product following an amplification reaction using, for example, a human's mucus sample and a primer system provided herein can indicate that that sample contains a virus (e.g., an influenza A virus, an adenovirus, a calicivirus, a coronavirus, a respiratory syncytial virus, or a SARS coronavirus).
  • the human can be diagnosed as being infected with the virus.
  • a mucus sample can be treated with a mucolytic agent to liquefy mucus within a mucus sample.
  • Samples can be processed to concentrate the nucleic acid and render it in a form to facilitate successful PCR reactions. This includes, but is not limited to, common methods to disrupt bilipid membranes, such as the use of detergents, digestion of protein complexes, such as the use of proteinase K, and reduction of polymerase inhibitors through the use of selective affinity columns.
  • Commercial kits for purification of DNA, RNA, or total nucleic acid are readily available from, for example, Qiagen and Roche.
  • a sample can be processed using a Qiagen QIAmp Viral RNA Mini Kit.
  • RNA viruses can be accomplished by synthesizing cDNA from RNA sequence templates.
  • cDNA synthesis can be accomplished using standard methods using, for example, RNA-dependant DNA polymerases, such as reverse transcriptase.
  • Such reactions can be primed with random oligonucleotide sequences, such as random hexamers and octamers, or by sequence specific oligonucleotide primers, including the same primers used for the PCR reaction.
  • the cDNA synthesis can be performed in a separate reaction vessel from the subsequent PCR reaction (commonly referred to as two-step rtPCR) or in the same reaction vessel as the PCR reaction (commonly referred to as single-step rtPCR).
  • Purified DNA and cDNA samples can be pooled and added to a PCR master mix containing water, salt buffers, magnesium ions, nucleotide monomers (dATP, dCTP, dGTP and dTTP), native or engineered Thermus aquaticus DNA-dependant DNA polymerase, and an intercalating dye, such as Sybr Green or LC Green.
  • the master mix and sample can then be added to a single loading port of a micro fluidic card and dispersed to all the reaction wells using centrifugation.
  • the cards can then be scored to isolate and seal each reaction chamber prior to thermocycling.
  • the cards can be individually thermocycled using commodity block thermocyclers or many cards thermocycled simultaneously using air- or water-based thermocyclers such as the BioOven or the H2OBIT, respectively.
  • Positive PCR amplification reactions can be detected during thermocycling for quantitative or qualitative analysis (real time PCR) or after completion of thermocycling (qualitative end-point PCR). Signals can be detected through fluorescence-channel emission of substrate bound intercalating dyes using commodity real-time PCR capable PCR platforms or by end-point reads using microplate scanner platforms. Both types of platforms can be used for melting-point analysis for validation of positive signals.

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Abstract

La présente invention concerne des systèmes, dispositifs et procédés impliqués dans la génération de polymères détectables. Par exemple, des systèmes diagnostiques, des dispositifs diagnostiques, des systèmes d'amorce et des collections de systèmes d'amorce sont proposés.
PCT/US2007/081286 2006-10-12 2007-10-12 Dispositifs de génération de polymères détectables WO2008127363A2 (fr)

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US11/548,989 US7462456B2 (en) 2006-10-12 2006-10-12 Devices for generating detectable polymers
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US11/549,008 US7521189B2 (en) 2006-10-12 2006-10-12 Devices for generating detectable polymers
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US11/548,986 US20080090229A1 (en) 2006-10-12 2006-10-12 Devices for generating detectable polymers
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US20060017789A1 (en) * 2004-07-23 2006-01-26 David Fletcher Inkjet printer cartridge refilling machine and method of operation thereof
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US20050202414A1 (en) * 2001-11-15 2005-09-15 The Regents Of The University Of California Apparatus and methods for detecting a microbe in a sample
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