EP2291541A1 - Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques - Google Patents

Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques

Info

Publication number
EP2291541A1
EP2291541A1 EP09716939A EP09716939A EP2291541A1 EP 2291541 A1 EP2291541 A1 EP 2291541A1 EP 09716939 A EP09716939 A EP 09716939A EP 09716939 A EP09716939 A EP 09716939A EP 2291541 A1 EP2291541 A1 EP 2291541A1
Authority
EP
European Patent Office
Prior art keywords
beads
dsdna
size
pna
specific
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.)
Withdrawn
Application number
EP09716939A
Other languages
German (de)
English (en)
Other versions
EP2291541A4 (fr
Inventor
Wan Wen Su
Jeffry Golden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Clean Earth Technologies LLC
Original Assignee
Clean Earth Technologies LLC
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
Application filed by Clean Earth Technologies LLC filed Critical Clean Earth Technologies LLC
Publication of EP2291541A1 publication Critical patent/EP2291541A1/fr
Publication of EP2291541A4 publication Critical patent/EP2291541A4/fr
Withdrawn legal-status Critical Current

Links

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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention relates to a series of DNA amplification and hybridization for the detection of multiple biological entities.
  • nucleic acid technologies include multiplexed real-time Polymerase Chain Reaction (PCR) using molecular beacons or TaqMan® assay [Wittwer, 2001] and multiplexed PCR coupled with microarray [Nosarabadi, US 7,083951] aiming at many targets in a single reaction.
  • Normal multiplexed PCR has high sensitivity. This varies from 20 - 2000 organisms for each amplicon type.
  • the limitations in these methods are as follows.
  • the multiplex real-time PCR assay has a 5-dye limit because of current maximum instrumentation capacity. It can simultaneously detect only five deoxyribonucleic acid (DNA) markers.
  • microarrays are time consuming, require denaturing amplified DNA to hybridize to the probes, and require expensive dye-labeled primers.
  • the probe oligonucleotides have to compete with the template complementary single-strand DNA (ssDNA) to bind dye-labeled ssDNA fragments, thus decreasing the overall assay efficiency.
  • ssDNA template complementary single-strand DNA
  • Lawrence Livermore National Laboratory (LLNL) developed a multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents [Wilson, 2005] for rapid screening of environmental samples.
  • the liquid array employs beads embedded with different ratios of red and infrared fluorescent dyes and each PCR product sequence to be detected is assigned one set of uniquely dyed beads.
  • the beads are conjugated with their assigned probe, a reverse complementary internal PCR product sequence.
  • the biotin-labeled PCR products from a multiplexed PCR assay are allowed to hybridize to the bead set and are then labeled with a second reporter dye complex, Streptavidin-phycoerythrin (SAPE), for detection using an automated flow cytometer.
  • SAPE Streptavidin-phycoerythrin
  • PNA peptide nucleic acid
  • dsDNA denature double- strand DNA
  • PNA PNA complement labeling for the simulataneous detection of organism specific DNA that is associated with target organisms in a sample with multiple organisms possibly present.
  • the PNA peptide backbone is more flexible than the natural ribose-phosphate backbone present in DNA.
  • PNA is more stable and resistant to degradation by nucleases and proteinases. It not only binds to complementary ssDNA, but it also binds specifically to dsDNA. This provides unique characteristics to detect complementary dsDNA.
  • Flow cytometry is an analytical method that allows both the rapid measurement of scattered light for particle size determination and measurement of fluorescence emission produced by suitably illuminated particles.
  • the particles are suspended in liquid and produce signals when they pass through a beam of light individually. Because measurements of each particle are made separately, the results are a correlated set of each individual particle's characteristics.
  • An important analytical feature of flow cytometry is its ability to measure multiple particle parameters such as scattered light and fluorescence emission. Scattered light collected in the same direction as the incident light reflects cell size where as the fluorescence is dependent upon the presence of fluorochromes on particles.
  • a combination of light scattering and fluorescence is a powerful approach to detect multiple targets in one sample without the need for a separation step.
  • a probe that attaches to ssDNA is used to link to a fluorescent dye, e.g., a biotin-based probe with cy5 or cy3 dye, or PCR primer is used to link to cy5 dye.
  • dsDNA can directly bind to a fluorescent dye.
  • the invention is a method for detection of multiple biological agents such as bacteria, viruses, spores, molds, and mycoplasma.
  • the method comprises the three steps of (1) multiplexing PCR to amplify different DNA targets and to produce dsDNA products, (2) mixing the multiple PCR dsDNA products with beads of various sizes wherein each size bead has a specific complementary PNA label and a fluorescent staining dye, and whereby the mixing causes the PNA-labeled beads to bind specific dsDNA by complementary sequences, and the fluorescent dsDNA dye to combine with dsDNA, and (3) flow cytometry is then performed to count the number of beads of each size and to measure the intensity of fluorescence of the beads of each size.
  • in the third step instead of performing flow cytometric detection, size separation of the labeled beads with bound dsDNA is performed and then the fluorescent signal of each size-separated fraction is measured.
  • Fig. 1. is a schematic diagram showing the three steps of the method of the instant invention.
  • the present invention comprises a method for detection and analysis of multiple biological agents.
  • Figure 1 shows the three steps of the multiplexed PCR-coupled PNA-labeled-beads flow cytometric assay.
  • the first step multiplexed PCR using oligonucleotide primers is preformed to obtain amplification of different DNA targets and to produce dsDNA products.
  • the multiple PCR dsDNA products are mixed with the PNA labeled beads and a single fluorescent staining dye, wherein beads of various sized have been prepared with various PNA labels so that a specific PNA label is applied to all beads of a specific size, and the beads with a specific PNA-label will bind to specific dsDNA by complementary sequences, and the fluorescent dsDNA dye will combine with dsDNA.
  • flow cytometry is performed to count and record the size and number of beads of each size and to measure and record the fluorescence intensity of the beads of a given size.
  • PNA can bind ssRNA that may be present in the PCR sample, it will be in a very small amount compared to the concentration of dsDNA amplicons.
  • the method offers high specificity and sensitivity, and it provides reductions in time and cost.
  • Table 1 presents a comparison of differences and advantages between the instant invention and the LLNL method. Table 1. Com arison of the instant invention and the LLNL method.
  • PCR products can be from 80 bp to 1000 bp. In a preferred embodiment, the best range is 100 bp to 150 bp, which is very suitable for efficient capture by PNA-beads and is conducive to the generation of ample fluorescent signal.
  • the efficiency of capturing dsDNA by PNA depends on the length of PNA.
  • the length of PNA can. range from 15 bp to 100 bp. In a preferred embodiment, the range is 18 bp to 25 bp, which is sufficient to capture complementary ds DNA fragments.
  • the beads may be made of any of a variety of materials. There are many types of suitable, commercially available beads. Exemplary materials are plastic, glass, silica gel, silica, latex, and ceramic. In a preferred embodiment, the beads are made of polystyrene, polycarbonate, silica gel, latex or glass. The beads may be modified by the addition of a carboxyl group to the bead material. The beads may also be coated with a coating having desired chemical or physical properties such as anti-agglomeration, hydrophobicity, hydrophillicity, hygroscopicity, opacity, reflectance, and color.
  • the size of the beads i.e., the diameters of the various size beads, can be the range from 0.1 ⁇ m to 100 ⁇ m. In a preferred embodiment, the range is 1 ⁇ m to 30 ⁇ m.
  • nearly monodisperse bead size fractions can be obtained by separation techniques using graded screen sieves or by flow cytometric separation methods.
  • the population of beads has a distribution of sizes. Ideally, this distribution is partitioned into set of discrete size ranges (size "bins") so that the beads can be sorted by size and in the flow cytometric determination of size, beads can be accurately identified as being within a specific size "bin".
  • Flow cytometers have size resolution that typically is ⁇ 0.5 ⁇ m, so discrimination of bead bins with width greater than 1 ⁇ m is readily achieved. Further, if the bead sizes are selected so that the bins are separated by a gap of about 0.25 to 1 ⁇ m according to the resolution of the instrument, then identification of bead size is nearly error free.
  • the distribution function of bead size may be relatively "flat", i.e., a constant as a function of bead size, or it may be selected so that smaller or larger beads are present in greater number to improve detection or to adjust the selectivity of the method in the case where some agents or their multiplex PCR product may be more or less abundant. If it is anticipated or suspected that an agent and its corresponding multiplex PCR product are in relatively small abundance, i.e., comprise a rare class, then weighting the bead size distribution to have a greater number of smaller beads can yield a disproportionately larger signal for the rare class. Similarly, if an agent class is expected to be greatly abundant, then fewer beads associated with the abundant class so that its signal does not overwhelm the less abundant or rare classes.
  • an NH3 group of PNA can be covalently linked (bonded) with a COOH group of the bead material or coating. This link is very strong and will remain unbroken during the performance of the assay.
  • Fluorescent DNA dyes are essential for flow cytometric detection and measurements.
  • dsDNA dyes used for flow cytometry and other detection methods [e.g., see Glazer, US 5,312,921].
  • Useful dsDNA dyes include SYBR green 1, ethidium bromide, thiazole orange (TO) and its derivatives, and propidium iodide (PI) [see Cosa, et al. and also, Fei, et al., and also, Nygren et al.]. Many of these are commercially available.
  • dsDNA dyes are SYBR green I, ethidium bromide, thiazole orange (TO), and propidium iodide (PI) which can bind dsDNA and generate a large signal.
  • flow cytometric detection comprises the determination of bead size, the counting of beads by size, and the measurement of fluorescent intensity by size of the beads.
  • Each size of bead represents a specific targeted sequence that is associated with a corresponding biological agent because beads of a specific size were coated by the specific PNA, which specifically hybridizes with the specific PCR product generated in the multiplex PCR step.
  • thresholds may be set.
  • the fluorescent intensity for an individual bead is greater than a threshold value, then, this bead represents a positive count for the detection of the specific agent.
  • Thresholds can be set for upper and lower limits of bead size for each size "bin", so that discrimination of bead size is improved and incorrect assignment of a bead to a size "bin” can be minimized or avoided. Accumulation of the statistics of counting and fluorescent intensity for each bead size provides detection and can be used for the determination of the relative abundance of the detected agents. Size Separation and Fluorescent Measurement Detection
  • the third step instead of performing flow cytometric detection, size separation of the labeled beads with bound dsDNA is performed and then the fluorescent signal of each size-separated fraction is measured.
  • Physical size separation is readily accomplished by a set of sized or graded screens or sieves so that each successive screen captures a smaller size fraction than the preceding screen.
  • Backwashing or other commonly used means can be used to remove the captured size fraction prior to measurement of the fluorescent signal of that size fraction, or the fluorescent signal may be measured in place if the captured beads comprise an approximately single bead thick layer on the screen.
  • signals from individual beads can be measured.
  • Using multiple dsDNA dyes with specific dyes assigned to specific size fractions enables improved discrimination and simultaneous monitoring.
  • Two or more aliquots (the initial set of aliquots) of the multiplexed PCR dsDNA products can be mixed with a selected single size fraction or multiple selected size fractions of the bead size distribution for which specific PNA labels have been applied to selected size fractions.
  • selected dye can be applied to a given aliquot, and then, the size-selected PNA labeled beads with bound dsDNA can be separated from the unbound PCR dsDNA products.
  • the selected dyes are chosen so that their emission wavelength or excitation wavelength differ and are sufficiently separated from each other so that detection at two or more emission wavelengths can be performed simultaneously or detection at a given wavelength with two different and sequentially pulsed excitation wavelengths is performed either when flow cytometric detection or size separation/fluorescent detection is performed on the separated beads or on a mixture of the previously separated beads of the initial the set of aliquots.
  • SYBR green I which, typically, is excited by light with wavelength of approximately 450-520 nm (peak ⁇ 488 nm) and emits with wavelengths in the vicinity of 490-640 nm (peak ⁇ 522 nm)
  • ethidium bromide which, typically, is excited by ultraviolet light (280-330 nm, peak ⁇ 300 nm) and emits orange light (560-720 nm, peak ⁇ 600).

Abstract

Cette invention concerne un procédé de détection de plusieurs entités biologiques, par exemple des agents de guerre biologique et du terrorisme, et des pathogènes menaçant la santé publique, qui consiste à multiplexer la PCR pour amplifier différentes cibles à ADN et pour produire des produits ADNdb, puis mélanger les nombreux produits d’ADNdb de PCR avec des billes de tailles spécifiques qui sont marquées avec des marqueurs PNA complémentaires spécifiques et un colorant fluorescent de sorte que les billes à marquage PNA se fixent avec des ADNdb spécifiques par séquences complémentaires et que le colorant ADNdb fluorescent se combine avec l’ADNdb, puis soit effectuer une cytométrie en flux pour compter les billes par taille et déterminer l’intensité de fluorescence par taille, soit exécuter la séparation par taille de deux fractions de tailles de billes ou plus et la détection en mesurant les signaux fluorescents de deux fractions de tailles de billes ou plus.
EP09716939A 2008-03-05 2009-03-05 Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques Withdrawn EP2291541A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3395208P 2008-03-05 2008-03-05
PCT/US2009/001466 WO2009111072A1 (fr) 2008-03-05 2009-03-05 Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques

Publications (2)

Publication Number Publication Date
EP2291541A1 true EP2291541A1 (fr) 2011-03-09
EP2291541A4 EP2291541A4 (fr) 2011-06-22

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EP09716939A Withdrawn EP2291541A4 (fr) 2008-03-05 2009-03-05 Dosage par cytométrie en flux avec des billes à marquage pna et avec couplage pcr multiplexé pour la détection simultanée de plusieurs agents biologiques

Country Status (2)

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EP (1) EP2291541A4 (fr)
WO (1) WO2009111072A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102719564B (zh) * 2012-06-25 2013-09-25 广西壮族自治区兽医研究所 鸭i型肝炎病毒、鸭圆环病毒和番鸭细小病毒的三重pcr试剂盒及其应用
WO2021092795A1 (fr) * 2019-11-13 2021-05-20 李峰 Procédé et dispositif de détection d'acide nucléique

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US6449562B1 (en) * 1996-10-10 2002-09-10 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
WO2007024840A2 (fr) * 2005-08-22 2007-03-01 Critical Therapeutics, Inc. Methode de quantification d'acides nucleiques
US20070231803A1 (en) * 2006-03-31 2007-10-04 Jensen Mark A Multiplex pcr mixtures and kits containing the same

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EP0912766B2 (fr) * 1996-06-04 2011-12-14 University of Utah Research Foundation Controle de l'hybridation pendant la pcr
US6280946B2 (en) * 1998-08-07 2001-08-28 Boston Probes, Inc. PNA probes, probe sets, methods and kits pertaining to the universal detection of bacteria and eucarya
US6994971B1 (en) * 1999-10-08 2006-02-07 University Of Utah Research Foundation Particle analysis assay for biomolecular quantification
US20050214825A1 (en) * 2000-02-07 2005-09-29 John Stuelpnagel Multiplex sample analysis on universal arrays
US20040161767A1 (en) * 2002-06-28 2004-08-19 Baldwin Brett R. Detection and quantification of aromatic oxygenase genes by real-time PCR
US20040038385A1 (en) * 2002-08-26 2004-02-26 Langlois Richard G. System for autonomous monitoring of bioagents
US7381547B2 (en) * 2004-04-28 2008-06-03 Tzam Diagnostics, Llc Methods and compositions to detect bacteria using multiplex PCR

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US6449562B1 (en) * 1996-10-10 2002-09-10 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
WO2007024840A2 (fr) * 2005-08-22 2007-03-01 Critical Therapeutics, Inc. Methode de quantification d'acides nucleiques
US20070231803A1 (en) * 2006-03-31 2007-10-04 Jensen Mark A Multiplex pcr mixtures and kits containing the same

Non-Patent Citations (1)

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Title
See also references of WO2009111072A1 *

Also Published As

Publication number Publication date
EP2291541A4 (fr) 2011-06-22
WO2009111072A1 (fr) 2009-09-11

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