US20120171725A1 - Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets - Google Patents
Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets Download PDFInfo
- Publication number
- US20120171725A1 US20120171725A1 US13/210,331 US201113210331A US2012171725A1 US 20120171725 A1 US20120171725 A1 US 20120171725A1 US 201113210331 A US201113210331 A US 201113210331A US 2012171725 A1 US2012171725 A1 US 2012171725A1
- Authority
- US
- United States
- Prior art keywords
- seq
- amplification
- primers
- target
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- 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]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Definitions
- the invention relates generally to methods for amplifying nucleic acids. More specifically, the invention relates to methods for using the polymerase chain reaction to amplify multiple nucleic acid sequences.
- PCR polymerase chain reaction
- MDD molecular differential diagnostic
- Diagnostic testing of clinical samples to find one or more causative disease agents has, in the past, required that microorganisms be isolated and cultured. This may take days, however, and in many cases a diagnosis must be acted upon within hours if the patient's life is to be saved.
- Analysis of a single clinical sample to identify multiple organisms in order to determine which one(s) may be the causative agent(s) of disease is the desired method for MDD, and methods have been developed to better achieve that goal. For example, multiplex PCR methods have been developed to amplify multiple nucleic acids within a sample in order to produce enough DNA/RNA to enable detection and identification of multiple organisms. Multiplex PCR has disadvantages, however.
- each target in a multiplex PCR reaction requires its own optimal reaction conditions, so increasing the number of targets requires that the reaction conditions for each individual target are less than optimal.
- multiple sets of high-concentration primers in a system often generate primer dimmers or give non-specific, background amplification. This lack of specificity also requires the additional steps of post-PCR clean-up and multiple post-hybridization washes. Crowded primers reduce the amplification efficiency by requiring the available enzymes and consuming substrates. Differences in amplification efficiency may lead to significant discrepancies in amplicon yields. For example, some loci may amplify very efficiently, while others amplify very inefficiently or fail to amplify at all. This potential for uneven amplification also makes it difficult to impossible to accurately perform end-point quantitative analysis.
- One method utilizes nested gene-specific primers used at very low concentrations to enrich the targets during the initial PCR cycling. Later, common primers are used to amplify all the targets. The entire reaction is performed in one tube, no additional rounds of PCR are required, and it does not require specialized instruments but may instead be performed using regular thermal cyders. There are disadvantages to this method, however. For example, because a low concentration of primers is used to enrich the targets during the initial cycles, the sensitivity of the assay is ultimately decreased, the initial enrichment cycles require longer annealing time for each cycle, and the enzyme is more likely to be less efficient over the number of cycles required to amplify the target.
- the present invention relates to a method for amplifying nucleic acids to enable detection of those nucleic acids, the method comprising the steps of amplifying one or more target nucleic acids using high concentration, target-specific primers in a first amplification reaction, thereby producing at least one nucleic acid amplicon containing at least one common primer binding site; rescuing the at least one nucleic acid amplicon; and amplifying the at least one nucleic acid amplicon in a second amplification reaction utilizing common primers which bind to the at least one common primer binding site.
- One aspect of the invention utilizes nested target-specific primers.
- Target nucleic acids may comprise DNA and/or RNA, and may comprise DNA and/or RNA of viral, bacterial, and/or fungal origin, as well as genomic DNA and/or RNA of human or other animal origin. Amplification may be performed by polymerase chain reaction (PCR) and/or RT-PCR.
- the source of the target nucleic acids may be from one or more clinical, environmental, or food samples and the method may be used in a wide variety of ways, including, for example, clinical diagnosis, environmental sampling, plant testing, food safety analysis, detection of genetic disorders, and/or detection of disease conditions. The method may be used for human and/or veterinary medical diagnoses.
- FIG. 1 is an illustration of the method of the invention, where F o represents forward-out primers; F i represents forward-in primers with a forward common primer tag (binding sequence); C f represents a forward common primer; R i represents a reverse-in primer with reverse common primer tag (binding sequence); R o represents a reverse-out primer; C r represents a reverse common primer; F a represents an additional forward primer; and R a represents an additional reverse primer, with these primers being positioned generally as indicated.
- the inventor has developed a new method for amplifying nucleic acids that may be used to detect the presence, and relative amounts present, of nucleic acids from viruses, bacteria, fungi, plant and/or animal cells for the evaluation of medical, environmental, food, and other samples to identify microorganisms and other agents within those samples.
- the method will be referred to herein as amplicon rescue multiplex polymerase chain reaction (“arm-PCR”).
- arm-PCR amplicon rescue multiplex polymerase chain reaction
- PCR amplifications of target nucleic acids are performed sequentially in two different reaction systems.
- These systems may comprise separate columns, reaction containers, or sections of a chip, for example, containing the target nucleic acid(s), primers, enzymes, nucleotides (e.g., dNTPs) and buffers necessary to amplify the target nucleic acid(s) to produce amplicons.
- primers primers
- enzymes nucleotides
- buffers necessary to amplify the target nucleic acid(s) to produce amplicons.
- reaction system it is intended to describe an Eppendorf tube, reaction chamber, or other containment device into which the necessary primers, enzymes, nucleotides, buffers, and/or other reagents are placed in order to perform one or more cycles of at least one polymerase chain reaction.
- reaction system may therefore refer to the same reaction containment vessel, but a different component of reagents—particularly primers—for performing the desired amplification step.
- reaction containment vessel is intended to mean a tube, plate well, or other vessel having a sufficient internal volume to contain primers, enzymes, nucleotides, buffers, and/or other reagents necessary to provide a reaction system.
- the term “rescue” is intended to mean the separation of amplicons from at least a portion of the primers of the first amplification.
- PCR is intended to mean the polymerase chain reaction, and may include PCR and/or RT-PCR procedures.
- high-concentration, target-specific, nested primers are used to perform a target-specific first amplification procedure.
- Primers are chosen from known sequences of viruses, bacteria, fungi, and/or other targets for which identification using nucleic acid detection is desired, and are specific for those target nucleic acids and/or closely related target nucleic acids.
- Target-specific primers may be used to amplify one or more (and preferably multiple) target nucleic acids of bacterial, viral, fungal, and/or other origin, for example.
- Nested primer concentration may generally be between 5-50 pmol. As illustrated in FIG.
- selected primers are “tagged” with additional nucleotides to provide an additional sequence that is not specific for the target nucleic acid(s) so that amplification of the target nucleic acid with such a primer will also incorporate into the resulting amplicon a binding site for a common primer that, unlike a target-specific primer, may be used to further amplify unrelated target nucleic acid amplicons (see A and B in FIG. 1 ).
- Amplification is performed for approximately 10-15 cycles, the reaction is terminated, and the resulting amplicons are rescued from the reaction mix for use in a second, target-independent amplification procedure, comprising a polymerase chain reaction primed by common primers which will, in a relatively indiscriminate manner, provide amplification of unrelated nucleotide sequences represented by the variety of amplicons rescued from the target-specific reaction.
- Amplicon rescue is then performed to minimize or eliminate the primers of the first reaction, while providing amplicons for use in the second amplification using common primers.
- Amplicon rescue may be performed in a variety of ways. For example, a small sampling from the completed first amplification reaction may be taken to provide amplicons for the second amplification. When a small sample is taken, it provides sufficient numbers of amplicons for the second amplification, while significantly decreasing (e.g., diluting) the remaining numbers of primers of the first amplification.
- Amplicon rescue may also be performed by removing a significant portion of the contents of the reaction system of the first amplification and adding to the remaining contents the common primer(s) with the necessary enzyme(s), nucleotides, buffer(s), and/or other reagents to perform a second amplification utilizing the common primer(s) to amplify the rescued amplicons in a second reaction system.
- Separation techniques may also be utilized to rescue amplicons. Such techniques may rely on size differences between the primers and amplicons, on tags that have been attached to the amplicons, the primers, or both, or other methods known to those of skill in the art. Once separated, all of the rescued amplicons or a part of the rescued amplicons may be used in the second amplification.
- the second amplification is performed in a different reaction system, which may or may not utilize the same reaction containment vessel.
- the second amplification amplifies the rescued amplicons using fresh buffer, nucleotides, and common primer(s). Common primers are chosen to provide efficient amplification of the rescued amplicons to provide significant numbers of copies of those amplicons at the end of the second amplification.
- the inventor By separating the reactions into a first, target-specific primer-driven amplification and a second, target-independent common primer-driven amplification, the inventor has developed a method that will provide specificity through the use of target-specific primers to amplify only the kinds and numbers of nucleic acids present from a particular target, and sensitivity achieved by the use of nested primers, the high concentration of target-specific primers, and the use of the common primer(s) to provide non-specific (target-independent) amplification at higher copy numbers.
- Target nucleic acids may be isolated from their respective sources by various means known to those of skill in the art. Detection of amplicons produced by the method may also be performed by various means known to those of skill in the art, such as application of the amplicons from the second amplification step to a printed array for hybridization and detection. Common primer sequences may include any sequence that will effectively provide for efficient initiation of an amplification reaction. Such sequences, and methods for designing them, are known to those of skill in the art.
- primers chosen from among SEQ ID NO: 1 (5′-TTCTTAGCGTATTGGAGTCC-3′), SEQ ID NO:2 (5′-AATGTACAGTATTGCGTTTTG-3′), or a combination of both, provide exceptional results in the second amplification reaction.
- the invention also provides primer kits for PCR amplification of target nucleotides, such kits comprising primers chosen from among the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36
- a method for automation of the method may be provided where the amplifications, separation, and detection are performed using a “lab-on-chip” device in a closed system.
- a first, target-specific amplification may be performed in a first reaction system (PCR1), where nested, unlabeled, high-concentration target-specific primers may be pre-loaded, together with dNTPs, buffer and enzymes to perform the desired PCR or RT-PCR amplification.
- PCR1 first reaction system
- PCR1 first reaction system
- nested, unlabeled, high-concentration target-specific primers may be pre-loaded, together with dNTPs, buffer and enzymes to perform the desired PCR or RT-PCR amplification.
- unused primers may be separated from nucleotide amplicons using capillary electrophoresis by means of electrodes activated between the PCR1 (negative) and a waste chamber (positive) to separate the primers from the amplicons.
- the electrode in the waste chamber may be turned off and a second reaction system (PCR2) positively charged.
- PCR2 second reaction system
- the larger molecular weight amplicons may therefore migrate to the PCR2 chamber, where they are mixed with pre-loaded common primers and fresh enzymes, dNTPs and buffer.
- the PCR products may be electrophoretically moved to the detection chamber to be hybridized to probes covalently fixed onto beads, the position of the beads in an array representing specific molecular targets.
- Target detection may therefore be performed by imaging analysis, for example, where positive results may be indicated by bright beads, as amplicons products may be labeled with fluorescent dyes or other chemical/biochemical labels. Unused PCR products and primers may then be removed and deposited in the waste chamber.
- a PCR chip may comprise a first reaction system fluidly connected to both a waste reservoir and a second reaction system, the waste reservoir and second reaction system each additionally comprising at least one electrode, the electrodes comprising a means for separating amplicons produced from the first reaction system.
- the second reaction system may be fluidly connected to a hybridization and detection chamber, the hybridization and detection chamber comprising microspheres, or beads, arranged so that the physical position of the beads is an indication of a specific target polynucleotide's presence in the sampled analyzed by means of the chip.
- the chip may be pre-loaded with reagents, or the reagents may be added by the user.
- pre-loaded reagents may include nested, high-concentration target-specific primers, dNTPs, polymerase enzymes, and buffer(s) for a first reaction system.
- the second reaction system may be preloaded with common primers, dNTPs, buffer, and polymerase enzymes.
- a patient sample may be loaded into at least one first reaction system by injecting the sample through soft, rubber-like polydimethylsiloxane (PDMS) material covering all or a portion of the chip.
- PDMS polydimethylsiloxane
- the first series of PCR cycles may be performed for the first amplification, to amplify the target sequences and to incorporate common primer binding sequences into at least a portion of the resulting amplicons.
- Amplicon products from the first reaction system may then be separated by on-chip electrophoresis performed in the microfluidic channel, the first reaction system being fluidly connected to at least one second reaction system and at least one waste reservoir, each of the second reaction systems and waste reservoirs additionally comprising at least one electrode, the electrodes promoting movement of the amplicons and unused primers from the first amplification reaction to a second reaction system and a waste reservoir, respectively.
- Amplicons moved to a second PCR reaction system may then be then subjected to a second amplification using common primers to amplify amplicons into which at least one common primer binding site has been incorporated during the first amplification in the first reaction system.
- the PCR products may be moved by microfluidic electrophoresis from the second reaction system to at least one hybridization and detection chamber, a second reaction system being fluidly connected to at least one hybridization and detection chamber.
- Within the hybridization and detection chamber may be microspheres, or beads, forming an array, the physical position of the beads indicating the specific target for detection.
- a bead array may comprise from about 1 to about 200 targets, with each target being represented by from about 1 to about 100 beads. If a specific target is not represented by the appropriate primers in the first amplification reaction, a software mask may be used to cover the related beads so that they will not interfere with the analysis.
- the hybridization and detection chamber may be fluidly connected to at least one wash chamber and at least one detection chamber, the wash chamber comprising reagents to assist in the removal of unused, labeled, primers and probes to reduce background, and the detection chamber comprising reagents such as streptavidine-Quantum dots, or streptavidine-PE for labeling amplified DNA for imaging analysis.
- the method of the invention may also be performed using a standard or modified PCR thermocycler.
- nucleotides, buffers, and primers may be loaded into standard PCR tubes in a first thermocycler for the first amplification.
- the contents of the tube may be removed by manual or automated means for rescue of the amplicons, and the newly-isolated amplicons may be placed into a second amplification tube where buffers, nucleotides, and enzyme(s) are introduced in order to perform the second amplification in the first or a second thermocycler, the thermocycler being programmed to cycle the reaction through the appropriate temperatures for the desired lengths of time.
- cycling times and the number of cycles may vary and may be determined by those of skill in the art.
- nested primers appear to improve the binding affinity of the polymerase, producing significantly more amplicons during the first amplification reaction.
- These amplicons may be produced from a variety of target polynucleotides within the sample, using a high concentration of target-specific primers.
- By incorporating into at least a portion of the amplicons during the first amplification at least one binding site for at least one common primer it is then possible, during the second amplification, to even more significantly increase the number of amplicons produced as a result of the amplification process.
- Common primers are chosen for their binding affinity and capacity to prime amplification during the second amplification.
- this three-step method (1 st amplification step, amplicon rescue, 2 nd amplification step), it is therefore possible to increase both the specificity and the sensitivity of the PCR process for identifying one or more target organism(s) from a sample containing multiple organisms.
- the inventor has discovered that this method does significantly increase both specificity and sensitivity, when compared to previously-described PCR methods.
- Automating the amplification-separation-amplification process enables the identification of a significant number of targets within a period of 1-3 hours, and has been shown to be effective for amplifying target nucleic acids from multiple microorganisms within a period of 1.5 hours, allowing rapid identification of a possible causative agent of disease to allow immediate steps to be taken toward treatment, isolation, implementation of public health plans for limiting exposure to epidemic-causing disease agents, bioterror agents, etc.
- Samples may be prepared for the PCR reactions by various means known to those of skill in the art. These methods may be provided as instructions provided with PCR kits containing buffers and enzymes, for example, or instructions may be obtained from various journal or patent publications. Methods for handling samples prior to preparation for the PCR amplification steps are also known to those of skill in the art, and may vary depending upon the source of the sample.
- Enzymes used for the amplifications are commercially available and may include, for example, Qiagen Multiplex mix or Qiagen Hot Start mix. Buffers are also commercially available, as are nucleotides (dNTPs) and other reagents. Thermocyclers are manufactured by and distributed by a variety of companies including, for example, Applied Biosystems and Bio-Rad. PCR reagent kits may also be obtained from various sources, including, for example, Qiagen (Gaithersburg, Md.).
- the invention provides a method that is suitable for identifying a single microorganism or multiple microorganisms, for example, from a sample that may contain a variety of microorganisms.
- a sample may be obtained from a clinical specimen (e.g., blood, saliva, tissue), from an environmental sample (e.g., water, soil), from a food sample, or other source.
- a clinical specimen e.g., blood, saliva, tissue
- an environmental sample e.g., water, soil
- Microorganisms that may be identified may include various genera and species of bacteria, viruses, and other DNA and/or RNA-containing organisms.
- a method such as the Luminex xMAP® technology may be utilized, and the detection step may be incorporated into the automated system along with the amplifications so that the automated system accomplishes the first amplification, the amplicon rescue, the second amplification, and detection.
- the Luminex xMAP® system for example, microspheres in suspension provide solid support for probe binding, also known as a “liquid chip” or “suspension array.” With xMAP® technology, molecular reactions take place on the surface of color-coded microspheres. For each pathogen, target-specific capture probes may be covalently linked to a specific set of color-coded microspheres.
- Labeled PCR products are captured by the bead-bound capture probes in a hybridization suspension.
- a microfluidics system delivers the suspension hybridization reaction mixture to a dual-laser detection device.
- a red laser identifies each bead by its color-coding, while a green laser detects the hybridization signal associated with each bead.
- Software is used to collect the data and report the results in a matter of seconds. The data is reported in the form of mean fluorescence intensity (MFI).
- MFI mean fluorescence intensity
- the method described herein enables one of skill in the art to couple high-specificity, high-sensitivity amplification and detection into one automated system. Using such a system, it is possible to analyze one or more clinical samples in a shorter period of time with greater sensitivity than has previously been possible with existing systems.
- An arm-PCR reaction was designed to amplify and detect pathogens responsible for food-borne diseases.
- the target gene used for each pathogen is listed in Table 1 below.
- Primers generated for each target are listed in Table 2.
- SupF and SupR indicate common primer sequences. Common primer sequences forming the tag for target-specific primers are shown in bold letters.
- F o , F i , R i and R o indicate the nested primers for each amplification target, while the D oligo indicates the detection probe that hybridizes to a specific sequence within the amplicon.
- the probe is covalently linked to a color coded bead for detection with the Luminex xMAP® instrument.
- the template was diluted to 10 pg/ ⁇ l, 1 pg/ ⁇ l, 0.1 pg/ ⁇ l, 0.01 pg/ ⁇ l, and 0.001 pg/ ⁇ l.
- a Qiagen Multiplex PCR kit was used to prepare a sample containing 44 ⁇ l of Multiplex Mix, 5 ⁇ l of primer mix, and 1 ⁇ l of template. Cycling conditions were as follows:
- samples were added to Millipore columns with a molecular weight cut-off of 50 kd and spun for 11 minutes at 13 k RPM to remove a substantial portion of the primers (molecular weight generally below 30 kd), rescuing amplicons with a molecular weight generally above 70 kd on top of the filter.
- the column was flipped and spun in a new collection tube for approximately 30 seconds to recover the amplicon for the next round of amplification.
- Hybridization was performed using 5 ⁇ l of PCR product added to 35 ⁇ l of bead (microsphere) mix and allowed to hybridize at 52° C. for 10 minutes. After 10 minutes, 10 ⁇ l of SA-PE was added (2 ⁇ SA-PE, Genaco Biomedical Sciences, Inc., was diluted 1:2 with 1 ⁇ TMAC) to each sample and allowed to hybridize at 52° C. for 5 minutes. After 5 minutes, 120 ⁇ l of 52° C. stop buffer was added to each sample and the samples were analyzed using a Luminex200 machine.
- MFI mean fluorescence intensity
Abstract
Description
- This application is a divisional application from U.S. non-provisional application Ser. No. 12/418,532, filed Apr. 3, 2009, which claimed the benefit of priority of U.S. Provisional Patent Application No. 61/042,259, filed Apr. 3, 2008.
- The invention relates generally to methods for amplifying nucleic acids. More specifically, the invention relates to methods for using the polymerase chain reaction to amplify multiple nucleic acid sequences.
- The development of the polymerase chain reaction (PCR) enabled the use of DNA amplification for a variety of uses, including molecular diagnostic testing. There are challenges associated with the use of PCR for molecular differential diagnostic (MDD) assays, however. PCR utilizes specific primers or primer sets, temperature conditions, and enzymes. PCR reactions may easily be contaminated, primer binding may require different conditions for different primers, primers should be specific for a target sequence in order to amplify only that target sequence, etc. This has made it even more difficult to amplify multiple sequences from a single sample.
- Diagnostic testing of clinical samples to find one or more causative disease agents has, in the past, required that microorganisms be isolated and cultured. This may take days, however, and in many cases a diagnosis must be acted upon within hours if the patient's life is to be saved. Analysis of a single clinical sample to identify multiple organisms in order to determine which one(s) may be the causative agent(s) of disease is the desired method for MDD, and methods have been developed to better achieve that goal. For example, multiplex PCR methods have been developed to amplify multiple nucleic acids within a sample in order to produce enough DNA/RNA to enable detection and identification of multiple organisms. Multiplex PCR has disadvantages, however. For example, each target in a multiplex PCR reaction requires its own optimal reaction conditions, so increasing the number of targets requires that the reaction conditions for each individual target are less than optimal. Furthermore, multiple sets of high-concentration primers in a system often generate primer dimmers or give non-specific, background amplification. This lack of specificity also requires the additional steps of post-PCR clean-up and multiple post-hybridization washes. Crowded primers reduce the amplification efficiency by requiring the available enzymes and consuming substrates. Differences in amplification efficiency may lead to significant discrepancies in amplicon yields. For example, some loci may amplify very efficiently, while others amplify very inefficiently or fail to amplify at all. This potential for uneven amplification also makes it difficult to impossible to accurately perform end-point quantitative analysis.
- One method utilizes nested gene-specific primers used at very low concentrations to enrich the targets during the initial PCR cycling. Later, common primers are used to amplify all the targets. The entire reaction is performed in one tube, no additional rounds of PCR are required, and it does not require specialized instruments but may instead be performed using regular thermal cyders. There are disadvantages to this method, however. For example, because a low concentration of primers is used to enrich the targets during the initial cycles, the sensitivity of the assay is ultimately decreased, the initial enrichment cycles require longer annealing time for each cycle, and the enzyme is more likely to be less efficient over the number of cycles required to amplify the target.
- A need still exists for more sensitive, faster, and more efficient methods for amplifying DNA and/or RNA from multiple targets to promote rapid identification of those targets.
- The present invention relates to a method for amplifying nucleic acids to enable detection of those nucleic acids, the method comprising the steps of amplifying one or more target nucleic acids using high concentration, target-specific primers in a first amplification reaction, thereby producing at least one nucleic acid amplicon containing at least one common primer binding site; rescuing the at least one nucleic acid amplicon; and amplifying the at least one nucleic acid amplicon in a second amplification reaction utilizing common primers which bind to the at least one common primer binding site. One aspect of the invention utilizes nested target-specific primers. Target nucleic acids may comprise DNA and/or RNA, and may comprise DNA and/or RNA of viral, bacterial, and/or fungal origin, as well as genomic DNA and/or RNA of human or other animal origin. Amplification may be performed by polymerase chain reaction (PCR) and/or RT-PCR. The source of the target nucleic acids may be from one or more clinical, environmental, or food samples and the method may be used in a wide variety of ways, including, for example, clinical diagnosis, environmental sampling, plant testing, food safety analysis, detection of genetic disorders, and/or detection of disease conditions. The method may be used for human and/or veterinary medical diagnoses.
-
FIG. 1 is an illustration of the method of the invention, where Fo represents forward-out primers; Fi represents forward-in primers with a forward common primer tag (binding sequence); Cf represents a forward common primer; Ri represents a reverse-in primer with reverse common primer tag (binding sequence); Ro represents a reverse-out primer; Cr represents a reverse common primer; Fa represents an additional forward primer; and Ra represents an additional reverse primer, with these primers being positioned generally as indicated. - The inventor has developed a new method for amplifying nucleic acids that may be used to detect the presence, and relative amounts present, of nucleic acids from viruses, bacteria, fungi, plant and/or animal cells for the evaluation of medical, environmental, food, and other samples to identify microorganisms and other agents within those samples. The method will be referred to herein as amplicon rescue multiplex polymerase chain reaction (“arm-PCR”). In this method, PCR amplifications of target nucleic acids are performed sequentially in two different reaction systems. These systems may comprise separate columns, reaction containers, or sections of a chip, for example, containing the target nucleic acid(s), primers, enzymes, nucleotides (e.g., dNTPs) and buffers necessary to amplify the target nucleic acid(s) to produce amplicons. By using high concentration primers in the first amplification reaction and rescuing the amplicons formed during that reaction for use in a second amplification reaction in a different reaction system, the inventor has developed a method that increases sensitivity and specificity, decreases the time needed to produce a detectable result, and readily lends itself to automation.
- It is to be understood that the term “comprising,” as used herein, may be substituted with the terms “consisting essentially of” and “consisting of.” Where the term “reaction system” is used, it is intended to describe an Eppendorf tube, reaction chamber, or other containment device into which the necessary primers, enzymes, nucleotides, buffers, and/or other reagents are placed in order to perform one or more cycles of at least one polymerase chain reaction. A different “reaction system” may therefore refer to the same reaction containment vessel, but a different component of reagents—particularly primers—for performing the desired amplification step. A “reaction containment vessel” is intended to mean a tube, plate well, or other vessel having a sufficient internal volume to contain primers, enzymes, nucleotides, buffers, and/or other reagents necessary to provide a reaction system. The term “rescue” is intended to mean the separation of amplicons from at least a portion of the primers of the first amplification. “PCR” is intended to mean the polymerase chain reaction, and may include PCR and/or RT-PCR procedures.
- In the first step of the method, high-concentration, target-specific, nested primers are used to perform a target-specific first amplification procedure. Primers are chosen from known sequences of viruses, bacteria, fungi, and/or other targets for which identification using nucleic acid detection is desired, and are specific for those target nucleic acids and/or closely related target nucleic acids. Target-specific primers may be used to amplify one or more (and preferably multiple) target nucleic acids of bacterial, viral, fungal, and/or other origin, for example. Nested primer concentration may generally be between 5-50 pmol. As illustrated in
FIG. 1 , selected primers are “tagged” with additional nucleotides to provide an additional sequence that is not specific for the target nucleic acid(s) so that amplification of the target nucleic acid with such a primer will also incorporate into the resulting amplicon a binding site for a common primer that, unlike a target-specific primer, may be used to further amplify unrelated target nucleic acid amplicons (see A and B inFIG. 1 ). Amplification is performed for approximately 10-15 cycles, the reaction is terminated, and the resulting amplicons are rescued from the reaction mix for use in a second, target-independent amplification procedure, comprising a polymerase chain reaction primed by common primers which will, in a relatively indiscriminate manner, provide amplification of unrelated nucleotide sequences represented by the variety of amplicons rescued from the target-specific reaction. - Amplicon rescue is then performed to minimize or eliminate the primers of the first reaction, while providing amplicons for use in the second amplification using common primers. Amplicon rescue may be performed in a variety of ways. For example, a small sampling from the completed first amplification reaction may be taken to provide amplicons for the second amplification. When a small sample is taken, it provides sufficient numbers of amplicons for the second amplification, while significantly decreasing (e.g., diluting) the remaining numbers of primers of the first amplification. Amplicon rescue may also be performed by removing a significant portion of the contents of the reaction system of the first amplification and adding to the remaining contents the common primer(s) with the necessary enzyme(s), nucleotides, buffer(s), and/or other reagents to perform a second amplification utilizing the common primer(s) to amplify the rescued amplicons in a second reaction system. Separation techniques may also be utilized to rescue amplicons. Such techniques may rely on size differences between the primers and amplicons, on tags that have been attached to the amplicons, the primers, or both, or other methods known to those of skill in the art. Once separated, all of the rescued amplicons or a part of the rescued amplicons may be used in the second amplification.
- The second amplification is performed in a different reaction system, which may or may not utilize the same reaction containment vessel. The second amplification amplifies the rescued amplicons using fresh buffer, nucleotides, and common primer(s). Common primers are chosen to provide efficient amplification of the rescued amplicons to provide significant numbers of copies of those amplicons at the end of the second amplification.
- By separating the reactions into a first, target-specific primer-driven amplification and a second, target-independent common primer-driven amplification, the inventor has developed a method that will provide specificity through the use of target-specific primers to amplify only the kinds and numbers of nucleic acids present from a particular target, and sensitivity achieved by the use of nested primers, the high concentration of target-specific primers, and the use of the common primer(s) to provide non-specific (target-independent) amplification at higher copy numbers. Furthermore, the use of high-concentration primers in a first amplification, followed by amplicon rescue—particularly when amplicon rescue is performed by isolating a portion of the first amplification by either removing that portion and placing it into a new reaction system or by removing a significant portion of the first amplification and adding to that the necessary reagents to form a second reaction system for a second, target-independent amplification—lends itself to automation. Not only can these steps be performed within a relatively closed reaction system, which limits the possibility of contamination, but the combination of first amplification, amplicon rescue, and second amplification provided by the method produces a specific, sensitive detection method for multiple targets from multiple samples within a period of less than 2 hours.
- Target nucleic acids may be isolated from their respective sources by various means known to those of skill in the art. Detection of amplicons produced by the method may also be performed by various means known to those of skill in the art, such as application of the amplicons from the second amplification step to a printed array for hybridization and detection. Common primer sequences may include any sequence that will effectively provide for efficient initiation of an amplification reaction. Such sequences, and methods for designing them, are known to those of skill in the art. The inventor has discovered that primers chosen from among SEQ ID NO: 1 (5′-TTCTTAGCGTATTGGAGTCC-3′), SEQ ID NO:2 (5′-AATGTACAGTATTGCGTTTTG-3′), or a combination of both, provide exceptional results in the second amplification reaction.
- The invention also provides primer kits for PCR amplification of target nucleotides, such kits comprising primers chosen from among the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, and combinations thereof.
- One example of a method for automation of the method may be provided where the amplifications, separation, and detection are performed using a “lab-on-chip” device in a closed system. For example, a first, target-specific amplification may be performed in a first reaction system (PCR1), where nested, unlabeled, high-concentration target-specific primers may be pre-loaded, together with dNTPs, buffer and enzymes to perform the desired PCR or RT-PCR amplification. After the first amplification has been allowed to proceed for the desired number of cycles, unused primers may be separated from nucleotide amplicons using capillary electrophoresis by means of electrodes activated between the PCR1 (negative) and a waste chamber (positive) to separate the primers from the amplicons. Upon movement of the primers to the waste chamber, the electrode in the waste chamber may be turned off and a second reaction system (PCR2) positively charged. The larger molecular weight amplicons may therefore migrate to the PCR2 chamber, where they are mixed with pre-loaded common primers and fresh enzymes, dNTPs and buffer. After the second amplification is performed in PCR2, the PCR products (amplicons) may be electrophoretically moved to the detection chamber to be hybridized to probes covalently fixed onto beads, the position of the beads in an array representing specific molecular targets. Target detection may therefore be performed by imaging analysis, for example, where positive results may be indicated by bright beads, as amplicons products may be labeled with fluorescent dyes or other chemical/biochemical labels. Unused PCR products and primers may then be removed and deposited in the waste chamber.
- In some embodiments, a PCR chip may comprise a first reaction system fluidly connected to both a waste reservoir and a second reaction system, the waste reservoir and second reaction system each additionally comprising at least one electrode, the electrodes comprising a means for separating amplicons produced from the first reaction system. The second reaction system may be fluidly connected to a hybridization and detection chamber, the hybridization and detection chamber comprising microspheres, or beads, arranged so that the physical position of the beads is an indication of a specific target polynucleotide's presence in the sampled analyzed by means of the chip.
- The chip may be pre-loaded with reagents, or the reagents may be added by the user. In one embodiment, pre-loaded reagents may include nested, high-concentration target-specific primers, dNTPs, polymerase enzymes, and buffer(s) for a first reaction system. The second reaction system may be preloaded with common primers, dNTPs, buffer, and polymerase enzymes. Using the chip, for example, a patient sample may be loaded into at least one first reaction system by injecting the sample through soft, rubber-like polydimethylsiloxane (PDMS) material covering all or a portion of the chip. In the first reaction system, the first series of PCR cycles may be performed for the first amplification, to amplify the target sequences and to incorporate common primer binding sequences into at least a portion of the resulting amplicons. Amplicon products from the first reaction system may then be separated by on-chip electrophoresis performed in the microfluidic channel, the first reaction system being fluidly connected to at least one second reaction system and at least one waste reservoir, each of the second reaction systems and waste reservoirs additionally comprising at least one electrode, the electrodes promoting movement of the amplicons and unused primers from the first amplification reaction to a second reaction system and a waste reservoir, respectively. Amplicons moved to a second PCR reaction system may then be then subjected to a second amplification using common primers to amplify amplicons into which at least one common primer binding site has been incorporated during the first amplification in the first reaction system. Following completion of the desired amplification cycles in the second reaction system, the PCR products (amplicons) may be moved by microfluidic electrophoresis from the second reaction system to at least one hybridization and detection chamber, a second reaction system being fluidly connected to at least one hybridization and detection chamber. Within the hybridization and detection chamber may be microspheres, or beads, forming an array, the physical position of the beads indicating the specific target for detection. A bead array may comprise from about 1 to about 200 targets, with each target being represented by from about 1 to about 100 beads. If a specific target is not represented by the appropriate primers in the first amplification reaction, a software mask may be used to cover the related beads so that they will not interfere with the analysis. The hybridization and detection chamber may be fluidly connected to at least one wash chamber and at least one detection chamber, the wash chamber comprising reagents to assist in the removal of unused, labeled, primers and probes to reduce background, and the detection chamber comprising reagents such as streptavidine-Quantum dots, or streptavidine-PE for labeling amplified DNA for imaging analysis.
- The method of the invention may also be performed using a standard or modified PCR thermocycler. For example, nucleotides, buffers, and primers may be loaded into standard PCR tubes in a first thermocycler for the first amplification. The contents of the tube may be removed by manual or automated means for rescue of the amplicons, and the newly-isolated amplicons may be placed into a second amplification tube where buffers, nucleotides, and enzyme(s) are introduced in order to perform the second amplification in the first or a second thermocycler, the thermocycler being programmed to cycle the reaction through the appropriate temperatures for the desired lengths of time. It should be understood that cycling times and the number of cycles may vary and may be determined by those of skill in the art.
- The use of nested primers appears to improve the binding affinity of the polymerase, producing significantly more amplicons during the first amplification reaction. These amplicons may be produced from a variety of target polynucleotides within the sample, using a high concentration of target-specific primers. By incorporating into at least a portion of the amplicons during the first amplification at least one binding site for at least one common primer, it is then possible, during the second amplification, to even more significantly increase the number of amplicons produced as a result of the amplification process. Common primers are chosen for their binding affinity and capacity to prime amplification during the second amplification. By the use of this three-step method (1st amplification step, amplicon rescue, 2nd amplification step), it is therefore possible to increase both the specificity and the sensitivity of the PCR process for identifying one or more target organism(s) from a sample containing multiple organisms. The inventor has discovered that this method does significantly increase both specificity and sensitivity, when compared to previously-described PCR methods.
- Automating the amplification-separation-amplification process enables the identification of a significant number of targets within a period of 1-3 hours, and has been shown to be effective for amplifying target nucleic acids from multiple microorganisms within a period of 1.5 hours, allowing rapid identification of a possible causative agent of disease to allow immediate steps to be taken toward treatment, isolation, implementation of public health plans for limiting exposure to epidemic-causing disease agents, bioterror agents, etc.
- Samples may be prepared for the PCR reactions by various means known to those of skill in the art. These methods may be provided as instructions provided with PCR kits containing buffers and enzymes, for example, or instructions may be obtained from various journal or patent publications. Methods for handling samples prior to preparation for the PCR amplification steps are also known to those of skill in the art, and may vary depending upon the source of the sample.
- Enzymes used for the amplifications are commercially available and may include, for example, Qiagen Multiplex mix or Qiagen Hot Start mix. Buffers are also commercially available, as are nucleotides (dNTPs) and other reagents. Thermocyclers are manufactured by and distributed by a variety of companies including, for example, Applied Biosystems and Bio-Rad. PCR reagent kits may also be obtained from various sources, including, for example, Qiagen (Gaithersburg, Md.).
- The invention provides a method that is suitable for identifying a single microorganism or multiple microorganisms, for example, from a sample that may contain a variety of microorganisms. Such a sample may be obtained from a clinical specimen (e.g., blood, saliva, tissue), from an environmental sample (e.g., water, soil), from a food sample, or other source. Microorganisms that may be identified may include various genera and species of bacteria, viruses, and other DNA and/or RNA-containing organisms.
- For identification of microorganisms, a method such as the Luminex xMAP® technology may be utilized, and the detection step may be incorporated into the automated system along with the amplifications so that the automated system accomplishes the first amplification, the amplicon rescue, the second amplification, and detection. In the Luminex xMAP® system, for example, microspheres in suspension provide solid support for probe binding, also known as a “liquid chip” or “suspension array.” With xMAP® technology, molecular reactions take place on the surface of color-coded microspheres. For each pathogen, target-specific capture probes may be covalently linked to a specific set of color-coded microspheres. Labeled PCR products are captured by the bead-bound capture probes in a hybridization suspension. A microfluidics system delivers the suspension hybridization reaction mixture to a dual-laser detection device. A red laser identifies each bead by its color-coding, while a green laser detects the hybridization signal associated with each bead. Software is used to collect the data and report the results in a matter of seconds. The data is reported in the form of mean fluorescence intensity (MFI).
- The method described herein enables one of skill in the art to couple high-specificity, high-sensitivity amplification and detection into one automated system. Using such a system, it is possible to analyze one or more clinical samples in a shorter period of time with greater sensitivity than has previously been possible with existing systems.
- The invention may be further described by means of the following non-limiting examples:
- An arm-PCR reaction was designed to amplify and detect pathogens responsible for food-borne diseases. The target gene used for each pathogen is listed in Table 1 below.
-
TABLE 1 Target Organism Target Gene Escherichia coli (E. coli) rfbE E. coli eac Salmonella invA Campylobacter jejuni (1) and coli (2) ceuE Shigella ipaH Yersinia enterocolitica yst Vibrio cholerae OMPW E. coli (ETEC) heat labile toxin LT E. coli (STEC) shiga toxin Stx Vibrio cholerae - cholera toxin Ctx E. coli (ETEC) heat stable toxin ST Vibrio parahaemolyticus tlh - Primers generated for each target are listed in Table 2. SupF and SupR indicate common primer sequences. Common primer sequences forming the tag for target-specific primers are shown in bold letters. Fo, Fi, Ri and Ro indicate the nested primers for each amplification target, while the D oligo indicates the detection probe that hybridizes to a specific sequence within the amplicon. The probe is covalently linked to a color coded bead for detection with the Luminex xMAP® instrument.
-
TABLE 2 Primer SEQ ID Name Primer Sequence NO: Sup F TTCTTAGCGTATTGGAGTCC 1 Sup R /5Biosg/AATGTACAGTATTGCGTTTTG 2 ceuE Fo CAACAAGTTGATTTTGAAGC 3 ceuE Fi TTCTTAGCGTATTGGAGTCCATTAATGCTTTAAAACCTGATC 4 ceuE Ri AATGTACAGTATTGCGTTTTGTTAAAAAATTTGCATTATCAAG ceue Ro ACCATAAAGTTTTGCAACGC 6 ceuE D1 /5AmMC12/CTC CAA CTT TAT TTG TAG ceuE2 Fo CAACAAGTTGATTTTGAAGC ceuE2 Fi TTCTTAGCGTATTGGAGTCCATTAATGCTTTAAAACCTGATC ceuE2 Ri AATGTACAGTATTGCGTTTTGTTAAAAAATTTGCATTATCAAG 10 ceuE2 Ro ACCATAAAGTTTTGCAACGC 11 ceuE D2 /5AmMC12/CTC CAA CTA TGT TTG TAG 12 rfbE2 Fo AGGATTAGCTGTACATAGGC 13 rfbE2 Fi TTCTTAGCGTATTGGAGTCCGGCATGACGTTATAGGCTAC 14 rfbE2 Ri AATGTACAGTATTGCGTTTTGTGTTCTAACTGGGCTAATCC 15 rfbE2 Ro CGTGATATAAAATCATCAGC 16 rfbE2 D /5AmMC12/GACAAATATCTGCGCTGCTAT 17 eac1 Fo CGATTACGCGAAAGATACCG 18 eac1 Fi TTCTTAGCGTATTGGAGTCCCCAGGCTTCGTCACAGTTGC 19 eac1 Ri AATGTACAGTATTGCGTTTTGCCAGTGAACTACCGTCAAAG 20 eac1 Ro TTTTCGGAATCATAGAACGG 21 eac1 D /5AmMC12/TTATGGAACGGCAGAGGTTA 22 invA1 Fo AACAGTGCTCGTTTACGACC 23 invA1 Fi TTCTTAGCGTATTGGAGTCCCTGGTACTAATGGTGATGATC 24 invA1 Ri AATGTACAGTATTGCGTTTTGGCGATCAGGAAATCAACCAG 25 invA1 Ro TGTAGAACGACCCCATAAAC 26 invA1 D /5AmMC12/TCGTCATTCCATTACCTACC 27 ipaH2 Fo GGATTCCGTGAACAGGTCGC 28 ipaH2 Fi TTCTTAGCGTATTGGAGTCCGCATGGCTGGAAAAACTCAG 29 ipaH2 Ri AATGTACAGTATTGCGTTTTGTCAGTGGCATCAGCAGCAAC 30 ipaH2 Ro CGCGACACGGTCCTCACAGC 31 ipaH2 D /5AmMC12/AGCTTCGACAGCAGTCTTTC 32 yst Fo GAAAAAGATAGTTTTTGTTC 33 yst Fi TTCTTAGCGTATTGGAGTCCATGCTGTCTTCATTTGGAGC 34 yst Ri AATGTACAGTATTGCGTTTTGGTGTCGATAATGCATCACTG 35 yst Ro CTTGTATACCTCAGCGGTTA 36 yst D /5AmMC12/CGGCCAAGAAACAGTTTCAG 37 ompW Fo CAAGTTTGTGTGATTTTTGTG 38 ompW Fi TTCTTAGCGTATTGGAGTCCCACAAAGATAACAACATAGCCC 39 ompW Ri AATGTACAGTATTGCGTTTTGTACGGCTAGGCAAATGGTTT 40 ompW Ro GTGAGCAAATACAGGAGCGG 41 ompW D1 /5AmMC12/AGGAAAACGTCATGAAAC 42 ompW2 Fo GTGAGTTGGCAGTTAATAGC 43 ompW2 Fi TTCTTAGCGTATTGGAGTCCGGTTAACGCTTGGCTATATG 44 ompW2 Ri AATGTACAGTATTGCGTTTTGGTAGAAATCTTATGTGAAAA 45 ompW2 Ro CTACCTAACTCACCACCAGA 46 ompW D2 /5AmMC12/CTGACAACATCAGTTTTG 47 LT1 Fo TCGATAGAGGAACTCAAATG 48 LT1 Fi TTCTTAGCGTATTGGAGTCCTCTTTATGATCACGCGAGAG 49 LT1 Ri AATGTACAGTATTGCGTTTTGGAAACATATCCGTCATCATA 50 LT1 Ro CTTCTCAAACTAAGAGAAGT 51 LT1 D /5AmMC12/GAACACAAACCGGCTTT 52 LT2 Fo TATGTTTAATGTTAATGATG 53 LT2 Fi TTCTTAGCGTATTGGAGTCCATACAGCCCTCACCCATATG 54 LT2 Ri AATGTACAGTATTGCGTTTTGCTGAGAATATGGTATTCCAC 55 LT2 Ro CCAAAATTAACACGATACCA 56 LT2 D /5AmMC12/AGGAGGTTTCTGCGTTA 57 stx Fo CATATATCTCAGGGGACCAC 58 stx Fi TTCTTAGCGTATTGGAGTCCGTGTCTGTTATTAACCACAC 59 stx Ri AATGTACAGTATTGCGTTTTGGTCAAAACGCGCCTGATAGA 60 stx Ro TTATTTTGCTCAATAATCAG 61 stx D /5AmMC12/GGGCAGTTATTTTGCTG 62 stx2 Fo ACAACGGTTTCCATGACAAC 63 stx2 Fi TTCTTAGCGTATTGGAGTCCGGACAGCAGTTATACCACTC 64 stx2 Ri AATGTACAGTATTGCGTTTTGGAAACCAGTGAGTGACGACT 65 stx2 Ro CCATTAACGCCAGATATGAT 66 stx2 D /5AmMC12/ACGTTCCGGAATGCAAAT 67 ctx1 Fo CAGATTCTAGACCTCCTGATG 68 ctx1 Fi TTCTTAGCGTATTGGAGTCCAGCAGTCAGGTGGTCTTATG 69 ctx1 Ri AATGTACAGTATTGCGTTTTGCATTTGAGTACCTCGGTCAA 70 ctx1 Ro CTTGCATGATCATAAAGGTTG 71 ctx1 D /5AmMC12/AGAGGACAGAGTGAGTAC 72 ctx2 Fo GGGCTACAGAGATAGATATTAC 73 ctx2 Fi TTCTTAGCGTATTGGAGTCCAGATATTGCTCCAGCAGCAG 74 ctx2 Ri AATGTACAGTATTGCGTTTTGCATGATGAATCCACGGCTCT 75 ctx2 Ro CGATGATCTTGGAGCATTCC 76 ctx2 D /5AmMC12/TATGGATTGGCAGGTTTC 77 ST Fo CTTTTTCACCTTTCGCTCAG 78 ST Fi TTCTTAGCGTATTGGAGTCCGATGCTAAACCAGCAGGGTC 79 ST Ri AATGTACAGTATTGCGTTTTGCAATTCACAGCAGTAATTGC 80 ST Ro CCGGTACAAGCAGGATTACA 81 ST D /5AmMC12/AGTAGTCCTGAAAGCATG 82 tlh1 Fo GATTCGTTTGACGGACGCAG 83 tlh1 Fi TTCTTAGCGTATTGGAGTCCCATGTTGATGACACTGCCAG 84 tlh1 Ri AATGTACAGTATTGCGTTTTGCGATCTCTTCTTGTGTTGAG 85 tlh1 Ro CAAGCACTTTCGCACGAATT 86 tlh1 D /5AmMC12/AAAGCGCCTCAGTTTAAG 87 tlh2 Fo AAGAGCACGGTTTCGTGAAC 88 tlh2 Fi TTCTTAGCGTATTGGAGTCCGACATCAACCGCTCATCGTC 89 tlh2 Ri AATGTACAGTATTGCGTTTTGCAGAACACAAACTTCTCAGC 90 tlh2 Ro CGGTGAGTTGCTGTTGTTGG 91 tlh2 D /5AmMC12/ATGTACACCCACGCATTG 92 - A primer mix containing 10 pmol of each of the Fo, Fi, Ri and Ro primers in Table 2. For this amplification, only one target template, Campylobacter jejuni, was included. The template was diluted to 10 pg/μl, 1 pg/μl, 0.1 pg/μl, 0.01 pg/μl, and 0.001 pg/μl. A Qiagen Multiplex PCR kit was used to prepare a sample containing 44 μl of Multiplex Mix, 5 μl of primer mix, and 1 μl of template. Cycling conditions were as follows:
-
- 95° C. for 15 minutes
- 94° C. for 15 seconds
- 55° C. for 15 seconds
- 72° C. for 15 seconds
- These three cycles repeated, 2-20 times (15 times total for this example.)
- 94° C. for 15 seconds
- 70° C. for 15 seconds
- These two cycles repeated, 6 times total for this example.
- 72° C. for 3 minutes
- 4° C. hold
- Upon completion of the first amplification as described above, samples were added to Millipore columns with a molecular weight cut-off of 50 kd and spun for 11 minutes at 13 k RPM to remove a substantial portion of the primers (molecular weight generally below 30 kd), rescuing amplicons with a molecular weight generally above 70 kd on top of the filter. The column was flipped and spun in a new collection tube for approximately 30 seconds to recover the amplicon for the next round of amplification.
- 10 μl of sample from the collection tube was added to 15 μl of Multiplex Mix, 1 μl of common primers, 10 pmol for the forward common primer and 40 pmol for the reverse common primer, and 14 μl of H2O. Samples were then placed in a thermocycler (Applied Biosystems, Foster City, Calif.) and run through the following cycles:
- 95° C. for 15 minutes (to heat activate the enzyme)
- 94° C. for 15 seconds
- 55° C. for 15 seconds×30 cycles
- 72° C. for 15 seconds
- 72° C. for 3 minutes
- 4° C. hold
- Hybridization was performed using 5 μl of PCR product added to 35 μl of bead (microsphere) mix and allowed to hybridize at 52° C. for 10 minutes. After 10 minutes, 10 μl of SA-PE was added (2× SA-PE, Genaco Biomedical Sciences, Inc., was diluted 1:2 with 1× TMAC) to each sample and allowed to hybridize at 52° C. for 5 minutes. After 5 minutes, 120 μl of 52° C. stop buffer was added to each sample and the samples were analyzed using a Luminex200 machine.
- Mean fluorescence intensity (MFI) numbers for arm-PCR and tem-PCR reactions are shown in Table 3.
-
TABLE 3 Template High concentration Low concentration Concentration nested primers - MFI nested primers - MFI 10 pg/μl 693 999 1 pg/μl 575 633 0.1 pg/μl 573 281 0.01 pg/μl 430 126 0.001 pg/μl 298 68 Blank 64 59 - These results indicate that although the signal is higher at high template concentrations when low concentration nested primers are used, the sensitivity of the high concentration nested primer method is about two logs higher. If the positive signal cutoff is 250 MFI, for example, this method can detect as little as 0.001 pg/μl, while the results of method previously described in the art for low concentration nested primers are negative between 0.1 pg/μl and 0.01 pg/μl. The time required for the entire process is approximately 210 minutes when using low concentration nested primers and approximately 150 minutes when using high concentration nested primers.
Claims (7)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/210,331 US20120171725A1 (en) | 2008-04-03 | 2011-08-15 | Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets |
US17/732,384 US20220251618A1 (en) | 2008-04-03 | 2022-04-28 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US4225908P | 2008-04-03 | 2008-04-03 | |
US12/418,532 US7999092B2 (en) | 2008-04-03 | 2009-04-03 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
US13/210,331 US20120171725A1 (en) | 2008-04-03 | 2011-08-15 | Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/418,532 Division US7999092B2 (en) | 2008-04-03 | 2009-04-03 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/732,384 Continuation US20220251618A1 (en) | 2008-04-03 | 2022-04-28 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
Publications (1)
Publication Number | Publication Date |
---|---|
US20120171725A1 true US20120171725A1 (en) | 2012-07-05 |
Family
ID=41133627
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/418,532 Active US7999092B2 (en) | 2008-04-03 | 2009-04-03 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
US13/210,331 Abandoned US20120171725A1 (en) | 2008-04-03 | 2011-08-15 | Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets |
US17/732,384 Pending US20220251618A1 (en) | 2008-04-03 | 2022-04-28 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/418,532 Active US7999092B2 (en) | 2008-04-03 | 2009-04-03 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/732,384 Pending US20220251618A1 (en) | 2008-04-03 | 2022-04-28 | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets |
Country Status (17)
Country | Link |
---|---|
US (3) | US7999092B2 (en) |
EP (1) | EP2271767B1 (en) |
JP (2) | JP5811483B2 (en) |
CN (3) | CN107385040B (en) |
AU (1) | AU2009231582B2 (en) |
BR (1) | BRPI0911082A2 (en) |
CY (1) | CY1117891T1 (en) |
DK (1) | DK2271767T3 (en) |
ES (1) | ES2586457T3 (en) |
HK (1) | HK1245846A1 (en) |
HR (1) | HRP20160923T1 (en) |
HU (1) | HUE029914T2 (en) |
LT (1) | LT2271767T (en) |
PL (1) | PL2271767T3 (en) |
PT (1) | PT2271767T (en) |
SI (1) | SI2271767T1 (en) |
WO (1) | WO2009124293A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014124451A1 (en) * | 2013-02-11 | 2014-08-14 | Cb Biotechnologies, Inc. | Method for evaluating an immunorepertoire |
US20170088895A1 (en) * | 2015-09-29 | 2017-03-30 | iRepertoire, Inc. | Immunorepertoire normality assessment method and its use |
WO2018165593A1 (en) * | 2017-03-09 | 2018-09-13 | iRepertoire, Inc. | Dimer avoided multiplex polymerase chain reaction for amplification of multiple targets |
US10529442B2 (en) * | 2015-03-06 | 2020-01-07 | iRepertoire, Inc. | Method for measuring a change in an individual's immunorepertoire |
Families Citing this family (47)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9424392B2 (en) | 2005-11-26 | 2016-08-23 | Natera, Inc. | System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals |
US11111544B2 (en) | 2005-07-29 | 2021-09-07 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US10083273B2 (en) | 2005-07-29 | 2018-09-25 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US11111543B2 (en) | 2005-07-29 | 2021-09-07 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US10081839B2 (en) | 2005-07-29 | 2018-09-25 | Natera, Inc | System and method for cleaning noisy genetic data and determining chromosome copy number |
US8501449B2 (en) | 2007-12-04 | 2013-08-06 | Proteon Therapeutics, Inc. | Recombinant elastase proteins and methods of manufacturing and use thereof |
ES2549184T3 (en) | 2008-04-16 | 2015-10-23 | Cb Biotechnologies, Inc. | Method to evaluate and compare immunorepertories |
EP2854056A3 (en) | 2009-09-30 | 2015-06-03 | Natera, Inc. | Methods for non-invasive pre-natal ploidy calling |
US11939634B2 (en) | 2010-05-18 | 2024-03-26 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US10316362B2 (en) | 2010-05-18 | 2019-06-11 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11326208B2 (en) | 2010-05-18 | 2022-05-10 | Natera, Inc. | Methods for nested PCR amplification of cell-free DNA |
US9677118B2 (en) | 2014-04-21 | 2017-06-13 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11332785B2 (en) | 2010-05-18 | 2022-05-17 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
US11339429B2 (en) | 2010-05-18 | 2022-05-24 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
US11322224B2 (en) | 2010-05-18 | 2022-05-03 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
EP2572003A4 (en) | 2010-05-18 | 2016-01-13 | Natera Inc | Methods for non-invasive prenatal ploidy calling |
US20190010543A1 (en) | 2010-05-18 | 2019-01-10 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11332793B2 (en) | 2010-05-18 | 2022-05-17 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11408031B2 (en) | 2010-05-18 | 2022-08-09 | Natera, Inc. | Methods for non-invasive prenatal paternity testing |
BR112013016193B1 (en) | 2010-12-22 | 2019-10-22 | Natera Inc | ex vivo method to determine if an alleged father is the biological father of a unborn baby in a pregnant woman and report |
CA2824854A1 (en) | 2011-01-14 | 2012-07-19 | Cb Biotechnologies, Inc. | Immunodiversity assessment method and its use |
JP6153874B2 (en) | 2011-02-09 | 2017-06-28 | ナテラ, インコーポレイテッド | Method for non-invasive prenatal ploidy calls |
US11782053B2 (en) | 2011-09-28 | 2023-10-10 | iRepertoire, Inc. | Identification of antigen-specific adaptive immune responses using ARM-PCR and high-throughput sequencing |
WO2013065646A1 (en) | 2011-10-31 | 2013-05-10 | アークレイ株式会社 | Method for measuring abundance of genes |
US9121051B2 (en) | 2011-10-31 | 2015-09-01 | Arkray, Inc. | Method of determining the abundance of a target nucleotide sequence of a gene of interest |
US8911949B2 (en) | 2011-11-11 | 2014-12-16 | Icubate, Inc. | Systems and methods for performing amplicon rescue multiplex polymerase chain reaction (PCR) |
US10262755B2 (en) | 2014-04-21 | 2019-04-16 | Natera, Inc. | Detecting cancer mutations and aneuploidy in chromosomal segments |
US10577655B2 (en) | 2013-09-27 | 2020-03-03 | Natera, Inc. | Cell free DNA diagnostic testing standards |
EP3561075A1 (en) | 2014-04-21 | 2019-10-30 | Natera, Inc. | Detecting mutations in tumour biopsies and cell-free samples |
CN104388579B (en) * | 2014-12-16 | 2018-11-09 | 上海速芯生物科技有限公司 | A kind of Arm-PCR detection methods of genetically engineered soybean and its derived varieties |
CN104611457B (en) * | 2015-03-04 | 2019-05-10 | 上海速芯生物科技有限公司 | Arm-PCR primer design method for multiple target point gene magnification |
WO2016145578A1 (en) | 2015-03-13 | 2016-09-22 | Syz Cell Therapy Co. | Methods of cancer treatment using activated t cells |
EP3294906A1 (en) | 2015-05-11 | 2018-03-21 | Natera, Inc. | Methods and compositions for determining ploidy |
DE102015012691A1 (en) | 2015-09-28 | 2017-03-30 | Biotecon Diagnostics Gmbh | Method for the quantitative detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae |
US11485996B2 (en) | 2016-10-04 | 2022-11-01 | Natera, Inc. | Methods for characterizing copy number variation using proximity-litigation sequencing |
US10011870B2 (en) | 2016-12-07 | 2018-07-03 | Natera, Inc. | Compositions and methods for identifying nucleic acid molecules |
GB201704754D0 (en) | 2017-01-05 | 2017-05-10 | Illumina Inc | Kinetic exclusion amplification of nucleic acid libraries |
WO2019183924A1 (en) | 2018-03-30 | 2019-10-03 | Syz Cell Therapy Co. | Improved multiple antigen specific cell therapy methods |
WO2019196088A1 (en) | 2018-04-13 | 2019-10-17 | Syz Cell Therapy Co. | Methods of obtaining tumor-specific t cell receptors |
WO2019196087A1 (en) | 2018-04-13 | 2019-10-17 | Syz Cell Therapy Co. | Methods of cancer treatment using tumor antigen-specific t cells |
US11525159B2 (en) | 2018-07-03 | 2022-12-13 | Natera, Inc. | Methods for detection of donor-derived cell-free DNA |
CN109055500A (en) * | 2018-09-13 | 2018-12-21 | 中国人民解放军疾病预防控制所 | A kind of fluorescence ring mediated isothermal amplification method based on molecular beacon |
GB201815041D0 (en) | 2018-09-14 | 2018-10-31 | Scancell Ltd | Epitopes |
WO2020146740A1 (en) | 2019-01-10 | 2020-07-16 | Iovance Biotherapeutics, Inc. | System and methods for monitoring adoptive cell therapy clonality and persistence |
GB202005779D0 (en) | 2020-04-21 | 2020-06-03 | Scancell Ltd | Anti-tumour immune responses |
GB202018395D0 (en) | 2020-11-23 | 2021-01-06 | Scancell Ltd | Immunotherapy |
US20220290214A1 (en) * | 2021-03-11 | 2022-09-15 | Sanigen Co., Ltd. | Primer set for detecting food poisoning bacteria by using next-generation sequencing method and method for detecting food poisoning bacteria by using the primer set |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5629158A (en) * | 1989-03-22 | 1997-05-13 | Cemu Bitecknik Ab | Solid phase diagnosis of medical conditions |
US5693502A (en) * | 1990-06-11 | 1997-12-02 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand inhibitors to DNA polymerases |
US5882856A (en) * | 1995-06-07 | 1999-03-16 | Genzyme Corporation | Universal primer sequence for multiplex DNA amplification |
US6312913B1 (en) * | 2000-07-21 | 2001-11-06 | Incyte Genomics, Inc. | Method for isolating and characterizing nucleic acid sequences |
US6514706B1 (en) * | 1998-10-26 | 2003-02-04 | Christoph Von Kalle | Linear amplification mediated PCR (LAM PCR) |
US20060147959A1 (en) * | 2004-03-31 | 2006-07-06 | The General Hospital Corporation | Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments |
US7097980B2 (en) * | 1996-05-29 | 2006-08-29 | Cornell Research Foundation, Inc. | Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions |
WO2006110855A2 (en) * | 2005-04-12 | 2006-10-19 | 454 Life Sciences Corporation | Methods for determining sequence variants using ultra-deep sequencing |
US20060269934A1 (en) * | 2005-03-16 | 2006-11-30 | Applera Corporation | Compositions and methods for clonal amplification and analysis of polynucleotides |
US20070059700A1 (en) * | 2003-05-09 | 2007-03-15 | Shengce Tao | Methods and compositions for optimizing multiplex pcr primers |
US20070141575A1 (en) * | 2003-10-13 | 2007-06-21 | Genaco Biomedical Products, Inc. | Method and kit for primer based multiplex amplification of nucleic acids |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE69032778T2 (en) * | 1989-07-18 | 1999-07-29 | Shimadzu Corp | Process for examining food poisoning caused by microorganisms and reagent therefor |
US5763173A (en) * | 1990-06-11 | 1998-06-09 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand inhibitors to DNA polymerases |
US5874557A (en) * | 1990-06-11 | 1999-02-23 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand inhibitors to DNA polymerases |
EP1036846A3 (en) * | 1994-02-28 | 2000-10-18 | Shimadzu Corporation | Oligonucleotides for detecting bacteria and detection process |
WO2004104172A2 (en) * | 2003-05-15 | 2004-12-02 | Bioarray Solutions, Ltd. | Hybridization-mediated analysis of polymorphisms |
US20060141518A1 (en) * | 2004-03-24 | 2006-06-29 | Lao Kai Q | Detection of gene expression |
FR2860802B1 (en) * | 2004-07-28 | 2008-02-22 | Bertin Technologies Sa | METHOD FOR ENABLING CHIMILUMINESCENCE READING OF A CHIP |
KR100671501B1 (en) * | 2005-02-28 | 2007-01-19 | 삼성에버랜드 주식회사 | Primer for detecting food poisoning and method for rapid detection of food born pathogene |
KR101515821B1 (en) * | 2005-09-01 | 2015-04-30 | 오스다이어그나스틱스 피티와이 엘티디. | Methods for the amplification, quantitation and identification of nucleic acids |
JP2007274934A (en) * | 2006-04-04 | 2007-10-25 | Nippon Meat Packers Inc | Primer set and method for detecting food poisoning bacterium |
-
2009
- 2009-04-03 US US12/418,532 patent/US7999092B2/en active Active
- 2009-04-03 DK DK09727817.0T patent/DK2271767T3/en active
- 2009-04-03 EP EP09727817.0A patent/EP2271767B1/en active Active
- 2009-04-03 ES ES09727817.0T patent/ES2586457T3/en active Active
- 2009-04-03 CN CN201710627329.4A patent/CN107385040B/en active Active
- 2009-04-03 CN CN2009801189681A patent/CN102066573A/en active Pending
- 2009-04-03 CN CN201510250072.6A patent/CN104928335A/en active Pending
- 2009-04-03 WO PCT/US2009/039552 patent/WO2009124293A1/en active Application Filing
- 2009-04-03 JP JP2011503231A patent/JP5811483B2/en active Active
- 2009-04-03 BR BRPI0911082-8A patent/BRPI0911082A2/en not_active Application Discontinuation
- 2009-04-03 AU AU2009231582A patent/AU2009231582B2/en active Active
- 2009-04-03 HU HUE09727817A patent/HUE029914T2/en unknown
- 2009-04-03 LT LTEP09727817.0T patent/LT2271767T/en unknown
- 2009-04-03 SI SI200931497A patent/SI2271767T1/en unknown
- 2009-04-03 PT PT97278170T patent/PT2271767T/en unknown
- 2009-04-03 PL PL09727817T patent/PL2271767T3/en unknown
-
2011
- 2011-08-15 US US13/210,331 patent/US20120171725A1/en not_active Abandoned
-
2015
- 2015-09-04 JP JP2015174617A patent/JP2016013133A/en active Pending
-
2016
- 2016-07-21 HR HRP20160923TT patent/HRP20160923T1/en unknown
- 2016-08-09 CY CY20161100781T patent/CY1117891T1/en unknown
-
2018
- 2018-04-19 HK HK18105098.0A patent/HK1245846A1/en unknown
-
2022
- 2022-04-28 US US17/732,384 patent/US20220251618A1/en active Pending
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5629158A (en) * | 1989-03-22 | 1997-05-13 | Cemu Bitecknik Ab | Solid phase diagnosis of medical conditions |
US5693502A (en) * | 1990-06-11 | 1997-12-02 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand inhibitors to DNA polymerases |
US5882856A (en) * | 1995-06-07 | 1999-03-16 | Genzyme Corporation | Universal primer sequence for multiplex DNA amplification |
US7097980B2 (en) * | 1996-05-29 | 2006-08-29 | Cornell Research Foundation, Inc. | Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions |
US6514706B1 (en) * | 1998-10-26 | 2003-02-04 | Christoph Von Kalle | Linear amplification mediated PCR (LAM PCR) |
US6312913B1 (en) * | 2000-07-21 | 2001-11-06 | Incyte Genomics, Inc. | Method for isolating and characterizing nucleic acid sequences |
US20070059700A1 (en) * | 2003-05-09 | 2007-03-15 | Shengce Tao | Methods and compositions for optimizing multiplex pcr primers |
US20070141575A1 (en) * | 2003-10-13 | 2007-06-21 | Genaco Biomedical Products, Inc. | Method and kit for primer based multiplex amplification of nucleic acids |
US20060147959A1 (en) * | 2004-03-31 | 2006-07-06 | The General Hospital Corporation | Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments |
US20060269934A1 (en) * | 2005-03-16 | 2006-11-30 | Applera Corporation | Compositions and methods for clonal amplification and analysis of polynucleotides |
WO2006110855A2 (en) * | 2005-04-12 | 2006-10-19 | 454 Life Sciences Corporation | Methods for determining sequence variants using ultra-deep sequencing |
Non-Patent Citations (20)
Title |
---|
Aoyagi (PCR, in Molecular Biology Problem Solver: A Laboratory Guide, Edited by Alan S. Gerstein, 2001) * |
Belgrader et al. (A Multiplex PCR-Ligase Detection Reaction Assay for Human Identity Testing, GENOME SCIENCE & TECHNOLOGY, Volume 1, Number 2, 1996, pgs. 77-87) * |
Brownie et al. (The elimination of primer-dimer accumulation in PCR, Nucleic Acids Research, 1997, Vol. 25, No. 16 3235-3241) * |
Buck et al ("Design Strategies and Performance of Custom DNA Sequencing Primers" Biotechniques. 1999. 27(3): pages 528-536) * |
Dear (Single Molecule PCR - Basic Protocols and Applications, in PCR Technology, 2004, pgs. 245-57) * |
Dieffenbach et al. (General concepts for PCR primer design, Genome Res. 1993 3: S30-S37) * |
Grunenwald (Optimization of Polymerase Chain Reactions, in Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling, 2003) * |
Haff (Improved Quantitative PCR Using Nested Primers, in PCR Methods and Applications, 1994, pgs. 332-337) * |
Heath (Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low density lipoprotein receptor gene, J Med Genet 2000;37:272-280) * |
Henegariu et al. (Multiplex PCR: Critical Parameters and Step-by-Step Protocol, BioTechniques 23:504-511, September 1997) * |
Jou et al. (Single-Tube, Nested, Reverse Transcriptase PCR for Detection of Viable Mycobacterium tuberculosis, JOURNAL OF CLINICAL MICROBIOLOGY, May 1997, p. 1161-1165) * |
Kramer et al. (Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization, in Current Protocols in Molecular Biology (2001) 15.1.1-15.1.14, 2001) * |
Lam et al. (Rapid Multiplex Nested PCR for Detection of Respiratory Viruses, JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2007, p. 3631-3640) * |
Lin et al. (Multiplex genotype determination at a large number of gene loci, Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2582-2587, March 1996) * |
Puig et al. (Detection of Adenoviruses and Enteroviruses in Polluted Waters by Nested PCR Amplification, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 2963-2970) * |
Roux (Optimization and Troubleshooting in PCR, in PCR Methods and Applications, Cold Spring Harbor Laboratory, 1995) * |
Sachse (Specificity and Performance of Diagnostic PCR Assays, in Methods in Molecular Biology, vol. 216: PCR Detection of Microbial Pathogens: Methods and Protocols, 2003, pgs. 3-19) * |
Shuber et al. (A Simplified Procedure for Developing Multiplex PCRs, Genome Res. 1995 5: 488-493) * |
Sotlar et al. (Detection and Typing of Human Papillomavirus by E6 Nested Multiplex PCR, JOURNAL OF CLINICAL MICROBIOLOGY, July 2004, p. 3176-3184) * |
Yourno (A method for nested PCR with single closed reaction tubes, Genome Res. 1992 2: 60-65) * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014124451A1 (en) * | 2013-02-11 | 2014-08-14 | Cb Biotechnologies, Inc. | Method for evaluating an immunorepertoire |
CN105164277A (en) * | 2013-02-11 | 2015-12-16 | Cb生物技术公司 | Method for evaluating an immunorepertoire |
KR20150141939A (en) * | 2013-02-11 | 2015-12-21 | 씨비 바이오테크놀로지스, 인크. | Method for evaluating an immunorepertoire |
KR102228488B1 (en) | 2013-02-11 | 2021-03-15 | 씨비 바이오테크놀로지스, 인크. | Method for evaluating an immunorepertoire |
US10529442B2 (en) * | 2015-03-06 | 2020-01-07 | iRepertoire, Inc. | Method for measuring a change in an individual's immunorepertoire |
US20170088895A1 (en) * | 2015-09-29 | 2017-03-30 | iRepertoire, Inc. | Immunorepertoire normality assessment method and its use |
US11047011B2 (en) * | 2015-09-29 | 2021-06-29 | iRepertoire, Inc. | Immunorepertoire normality assessment method and its use |
WO2018165593A1 (en) * | 2017-03-09 | 2018-09-13 | iRepertoire, Inc. | Dimer avoided multiplex polymerase chain reaction for amplification of multiple targets |
CN110662756A (en) * | 2017-03-09 | 2020-01-07 | 艾瑞普特公司 | Multiplex polymerase chain reaction avoiding dimers for multi-target amplification |
EP3585797A4 (en) * | 2017-03-09 | 2020-12-30 | Irepertoire, Inc. | Dimer avoided multiplex polymerase chain reaction for amplification of multiple targets |
US20220259653A1 (en) * | 2017-03-09 | 2022-08-18 | iRepertoire, Inc. | Dimer avoided multiplex polymerase chain reaction for amplification of multiple targets |
Also Published As
Publication number | Publication date |
---|---|
US20090253183A1 (en) | 2009-10-08 |
SI2271767T1 (en) | 2016-12-30 |
PL2271767T3 (en) | 2017-01-31 |
CN107385040A (en) | 2017-11-24 |
LT2271767T (en) | 2016-09-26 |
CN107385040B (en) | 2022-02-15 |
JP5811483B2 (en) | 2015-11-11 |
AU2009231582B2 (en) | 2015-02-26 |
PT2271767T (en) | 2016-08-18 |
ES2586457T3 (en) | 2016-10-14 |
AU2009231582A1 (en) | 2009-10-08 |
WO2009124293A1 (en) | 2009-10-08 |
HK1245846A1 (en) | 2018-08-31 |
HRP20160923T1 (en) | 2016-10-21 |
DK2271767T3 (en) | 2016-08-29 |
EP2271767A1 (en) | 2011-01-12 |
US7999092B2 (en) | 2011-08-16 |
CN102066573A (en) | 2011-05-18 |
EP2271767A4 (en) | 2011-08-24 |
BRPI0911082A2 (en) | 2015-08-04 |
EP2271767B1 (en) | 2016-06-29 |
JP2016013133A (en) | 2016-01-28 |
US20220251618A1 (en) | 2022-08-11 |
HUE029914T2 (en) | 2017-04-28 |
JP2011516069A (en) | 2011-05-26 |
CN104928335A (en) | 2015-09-23 |
CY1117891T1 (en) | 2017-05-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220251618A1 (en) | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets | |
JP7110155B2 (en) | Multiplex pyrosequencing using non-interfering, noise-canceling polynucleotide identification tags | |
US9222126B2 (en) | Methods for point-of-care detection of nucleic acid in a sample | |
WO2011001496A1 (en) | Sample analysis method and assay kit for use in the method | |
EP2402462A1 (en) | Internally controlled multiplex detection and quantification of microbial nucleic acids | |
US9416398B2 (en) | Generic buffer for amplification | |
US9725754B2 (en) | Generic sample preparation | |
AU2005210362B8 (en) | Method of detecting nucleic acid and utilization thereof | |
WO2009087685A2 (en) | Method for detection of hepatitis nucleic acid and uses thereof | |
WO2012051504A2 (en) | Quantitative multiplexed identification of nucleic acid targets | |
US20160230208A1 (en) | Generic PCR | |
EP0517361A1 (en) | A method for detecting and identifying pathogenic organisms using target sequences as detectors | |
WO2022203008A1 (en) | Method and kit for identifying bacteria that cause sepsis | |
JP4528889B1 (en) | Sample analysis method and assay kit used therein | |
Point | 16.1 Polymerase chain reaction (PCR) | |
Schewe et al. | PCR-ELISA 24 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: HUDSON ALPHA INSTITUTE FOR BIOTECHNOLOGY, ALABAMA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HAN, JIAN;REEL/FRAME:036181/0976 Effective date: 20080529 |
|
AS | Assignment |
Owner name: CB BIOTECHNOLOGIES, INC., ALABAMA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HUDSON ALPHA INSTITUTE FOR BIOTECHNOLOGY;REEL/FRAME:036984/0819 Effective date: 20130617 |
|
AS | Assignment |
Owner name: IREPERTOIRE, INC., ALABAMA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CB BIOTECHONOLOGIES INCORPORATED;REEL/FRAME:043741/0040 Effective date: 20160627 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCV | Information on status: appeal procedure |
Free format text: NOTICE OF APPEAL FILED |
|
STCV | Information on status: appeal procedure |
Free format text: APPEAL BRIEF (OR SUPPLEMENTAL BRIEF) ENTERED AND FORWARDED TO EXAMINER |
|
STCV | Information on status: appeal procedure |
Free format text: EXAMINER'S ANSWER TO APPEAL BRIEF MAILED |
|
STCV | Information on status: appeal procedure |
Free format text: BOARD OF APPEALS DECISION RENDERED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |