WO2014018828A1 - Détection de produits d'amplification - Google Patents

Détection de produits d'amplification Download PDF

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WO2014018828A1
WO2014018828A1 PCT/US2013/052189 US2013052189W WO2014018828A1 WO 2014018828 A1 WO2014018828 A1 WO 2014018828A1 US 2013052189 W US2013052189 W US 2013052189W WO 2014018828 A1 WO2014018828 A1 WO 2014018828A1
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oligonucleotide
amplification
quencher
oligo
label
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PCT/US2013/052189
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Nathan TANNER
Yinhua Zhang
Thomas C. Evans
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New England Biolabs, Inc.
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    • 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
    • C12Q1/6851—Quantitative amplification

Definitions

  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • HDA helicase dependent amplification
  • ICAN isothermal and chimeric primer-initiated amplification of nucleic acids
  • LAMP strand-displacing DNA polymerase
  • LAMP uses 4 core primers (FIP, BIP, F3, and B3) recognizing 6 distinct sequence regions on the target ( Figure l(a)-(b)), with two primers containing sequence (F1C, B1C) that results in loop structures which facilitate exponential amplification (Notomi, et al., Nucleic Acids Res., 28 : E63
  • the use of multiple target sequence regions confers a high degree of specificity to the reaction.
  • Two additional primers termed loop primers, can be added to increase reaction speed, resulting in 6 total primers used per target sequence (Nagamine, et al., Mol. Cel. Probes, 16 : 223-9 (2002)).
  • the LAMP reaction rapidly generates amplification products as multimers of the target region in various sizes, and is substantial in total DNA synthesis (> 10 pg, >50x PCR yield) (Notomi, et al. (2000); Nagamine, et al., Clin. Chem. , 47 : 1742-3 (2001)) (see Figure l(a)-(b)) .
  • Measurement of LAMP amplification product may be performed using fluorescence detection of double-stranded DNA (dsDNA) with an intercalating or magnesium-sensitive fluorophore (Notomi, et al. (2000); Goto, et al., Biotechniques, 46: 167-72, (2009)), bioluminescence through pyrophosphate conversion (Gandelman, et al, PloS One, 5:el4155 (2010)), turbidity detection of precipitated magnesium pyrophosphate (Mori, et al., Biochem. Biophys. Methods 59 : 145-57 (2004); Mori, et al., Biochem. Biophys Res. Commun. , 289: 150-4 (2001)), or even visual examination through
  • PCR requires a pair of primers and thermophilic DNA polymerase such as Taq DNA polymerase. During amplification, cycles of denaturation, annealing and primer extension steps allow primers to bind to the target sequence and DNA synthesis.
  • thermophilic DNA polymerase such as Taq DNA polymerase.
  • endpoint or real time Two types of detection are commonly used : endpoint or real time.
  • a typical endpoint detection is agarose gel
  • the detection of qPCR can be divided into two types: the first type uses a double strand DNA intercalating dye and the second type uses a sequence specific probes.
  • a number of methods have been described using sequence-specific probe (Holland, et al. (1991); VanGuilder, et al. (2008); Didenko (2001); Bustin 2006)).
  • sequence-specific probe Holland, et al. (1991); VanGuilder, et al. (2008); Didenko (2001); Bustin 2006).
  • these typically require design of fluorescent probes in addition to the PCR primers.
  • a composition in a buffer, that includes: a first oligonucleotide comprising a primer sequence which is also a target sequence for priming an amplification reaction, the first
  • oligonucleotide having a quencher or fluorescent label having a quencher or fluorescent label
  • oligonucleotide having a sequence suitable for hybridizing to a portion of the first oligonucleotide under stringent conditions to form a stable duplex, the second oligonucleotide having a fluorescent label if the first oligonucleotide has a quencher label, or having a quencher label if the first oligonucleotide has a fluorescent label; and a third oligonucleotide comprising some or all of the primer sequence contained in the first oligonucleotide and not including a quenching or fluorescent label wherein the molar ratio of the first oligonucleotide to the third nucleotide is in the range of 2:8 to 8: 2.
  • compositions include one or more of the following features: the second oligonucleotide at a concentration that is substantially the same as the first oligonucleotide; the first oligonucleotide having a quencher label and the second oligonucleotide having a fluorescent label or the first oligonucleotide having a fluorescent label and the second oligonucleotide having a quencher label; the first oligonucleotide and the third oligonucleotide combined in at least 1 : 1 molar ratio; a strand
  • polymerases wherein one of the polymerases is an archeal polymerase.
  • a method for detecting an amplification product of a polynucleotide includes; adding to a
  • polynucleotide a first oligonucleotide comprising a primer sequence for priming the amplification from the polynucleotide template at a first location on the polynucleotide and having a quencher or fluorescent label; and a second oligonucleotide hybridized to the first oligonucleotide under stringent conditions to form a stable duplex and having a fluorescent label if the first oligonucleotide has a quencher label, or having a quencher label if the first oligonucleotide has a fluorescent label; and a third oligonucleotide
  • the primer sequence contained in the first oligonucleotide comprising some or all of the primer sequence contained in the first oligonucleotide and not including a quenching or fluorescent label wherein the molar ratio of the first oligonucleotide to the third nucleotide is in the range of 2 :8 to 8:2; permitting amplification of the polynucleotide; and detecting the amplified product of the polynucleotide.
  • Various embodiments of the method include additionally adding one or more of the following features: a strand displacement polymerase; a plurality of polymerases, wherein one of the polymerases is an archeal polymerase; a fourth oligonucleotide comprising some or all of a sequence for annealing to a priming site on a second location on the polynucleotide; and/or a fourth, fifth and sixth oligonucleotide where the fourth
  • oligonucleotide is unlabeled and competes with a dimerized labeled fifth and sixth oligonucleotide; an amount of the first oligonucleotide being X/N where X is the optimal primer concentration in a single-plex reaction and N is the number of different templates for which primer sets are present in the reaction mixture, and/or X is in the range of 0.1 ⁇ - 2 ⁇ .
  • Other features may optionally include one or more of the following: the first oligonucleotide having a quencher label and the second oligonucleotide having a fluorescent label, or the first oligonucleotide having a fluorescent label and the second oligonucleotide having a quencher label; and/or combining the first oligonucleotide and the third oligonucleotide in at least a 1 : 1 ratio.
  • Other features may include releasing the second oligonucleotide by means of primer extension from the fourth oligonucleotide.
  • Additional features may include amplifying DNA using PCR, reverse transcription PCR, LAMP, or reverse transcription LAMP; detecting an amplification product of multiple polynucleotides in a multiplex reaction mixture and optionally including an internal standard; and/or determining the size of the amplification product.
  • Additinoal features include the monitoring of amplification using pH sensitive dyes described in U.S. patent application number 13/799,995. Non-template amplification may be inhibited using the compositions and methods described in U.S. patent application number 13/799,463.
  • FIG. 1(a) and 1(b) schematically shows LAMP amplification using labeled oligonucleotides and non-labeled oligonucleotides for forward and backward strand displacement.
  • the LAMP reaction relies on a strand-displacing
  • Figure 1(a) shows 3 different oligonucleotides (Oligo 1 (labeled Flc-
  • F2 Oligo 2 (labeled Fl) and Oligo 3 (unlabeled Flc-F2)) identified as a forward internal primer (FIP) because of the association with one end of the target DNA (3' end).
  • the label is a 5'-quencher (black star) or 3'-fluorophore of Flc (white star).
  • the sequences of Oligo 1, 2 and 3 correspond to the target sequence at the 3' end as shown in Fig l(b)(i).
  • Figure l(b)(i-iv) shows LAMP amplification with the three forward oligonucleotides described in Figure 1(a) and a backward oligonucleotide (Oligo 4) (unlabelled Blc-B2) at the 5'end of the DNA.
  • a labeled Oligo 4 may be used with an Oligo 5/6;( B2-Blc/Bl) to further increase the detection signal.
  • Figure l(b)(i) shows initiation of amplification of a polynucleotide template.
  • Polymerase activity initiates from F2 which acts as a single strand primer having a double-stranded labeled tail which has a quenched fluorescent terminus.
  • polymerase initiates from F3 to "bump" the downstream product from initiation at F2.
  • LoopF and LoopB primers are not shown.
  • Figure 2(a)-(d) shows primer designs for LAMP and PCR amplification using labeled Oligo 1/2 and unlabeled Oligo 3 as defined in Figure 1(a) at the 5' end of the template polynucleotide.
  • Primer sequence complementary to the target polynucleotide is found in both Oligo 1 and 3 (F2).
  • Figure 2(a) and 2(c) the primer sequence is entirely complementary to the priming region in the target polynucleotide whereas in Figure 2(b) and 2(d) the primer is shown to have only partial complementarity with the priming sequence in the target polynucleotide where the F2 primer may extend 3' or 5' beyond the priming sequence as shown
  • Figure 2(a) shows LAMP primers where the primer is complementary to the priming region and where Oligo 3 is the same as oligo 1 but without LI.
  • Figure 2(b) shows LAMP primers where the primer is only partially complementary to the priming region and where Oligo 3 is the same as oligo 1 but without LI.
  • Figure 2(c) shows PCR primers where the primer is complementary to the priming region and where Oligo 3 corresponds to F2 only.
  • Figure 2(d) shows PCR primers where the primer is only partially complementary to the priming region and where Oligo 3 corresponds to F2 only.
  • Figure 3(a)-(c) shows that the amplification methods shown in Figures l(b)(i-iv) and Figure 2(a)-(b) can be used effectively in multiplex LAMP reactions even if the amplification rate varies for different polynucleotides having different fluorescent labels.
  • Relative fluorescent units normalized to maximum fluorescence (RFU) is plotted against time of the reaction
  • Figure 3(b) shows detection of E. coli DNA (dnaE) with Cy5-labeled Oligo 2 and human DNA (BRCA1) with Cy5.5-labeled Oligo 2, with dark quencher labeled Oligo 1.
  • Figure 3(c) shows maintenance of amplification for a single
  • concentration internal standard (82.5 ng C. elegans genomic DNA; lec-10 target; ROX Oligo 2) that is not influenced by and therefore independent of LAMP amplification of varying amounts of a test target DNA (10 pg - 100 ng HeLa genomic DNA; CFTR target; 6-FAM Oligo 2).
  • a test target DNA (10 pg - 100 ng HeLa genomic DNA; CFTR target; 6-FAM Oligo 2).
  • the relatively high concentration of the standard is amplified at a high rate while 10 pg of Hela genomic DNA is amplified significantly slower although the amplification rate is increased as expected with increasing concentration of Hela genomic DNA.
  • Figure 4(a)-(b) show single multiplex reactions consisting of 3 and 4 targets where the number of detection reactions in a multiplex reaction is limited only by the availability of distinguishable fluorescent tags and the number of channels in a fluorimenter or capillary electrophoresis device.
  • Figure 4(a) shows a triplex reaction that results in detection of three genomic DNAs: E. coli genomic DNA (Cy5 Oligo 2), lambda genomic DNA (HEX Oligo 1), and C. elegans genomic DNA (ROX Oligo 2). Concentration of each primer set was scaled by 1/3 for triplex reactions.
  • Figure 4(b) shows the detection of four genomic DNAs where three DNAs are the same as used in the triplex in Figure 4(a) and the fourth DNA is human genomic DNA (Cy5.5 Oligo 2).
  • the concentration of each primer set was scaled by 1/4 for quadruplex reactions. The same overall
  • reactions contained eight oligonucleotides per target and thirty-two total
  • Figure 5 shows effects of the stoichiometry of Oligos 1/2 and 3 on both amplification threshold time and signal amplitude in LAM P reactions performed with dnaE/Cy5 and 5 ng E. coli genomic DNA.
  • the X-axis is background-subtracted Cy5 signal from displacement of Oligo 2 and the y- axis is threshold time (Ct) of each reaction.
  • Use of 100% Oligo 1/2 or 75% Oligo 1/2 : 25% Oligo 3 resulted in high signal amplitude, but substantially increased threshold time.
  • FIG. 6(a)-(b) shows detection of PCR amplification using Oligos 1/2 and Oligo 3.
  • Realtime PCR was performed to detect the E. coli 16s rRNA gene.
  • the primers ( Figure 2, Table 2) contain regular PCR primers (F, Oligo 3, and R primers) at 200 ⁇ each and a pair of primers (D-F, Oligo 1 , and Fq, Oligo 2) at 80uM each.
  • Ten-fold serial dilutions of E. coli genomic DNA from 100 ng to 0. 1 pg (equivalent of 20xl0 6 to 20 copies) were used as template.
  • Figure 6(a) shows realtime fluorescence signal during PCR cycling .
  • Figure 6(b) shows the resulting standard curve, determined by the cycle number when the signal crossing the amplification threshold (Cq value, Y-axis) plotted against the log value of the copy number.
  • Figure 7(a)-(c) shows that both copy number and size determinations for C. elegans act-1 gene can be determined using a 10-fold dilution series of genomic DNA (approximately 760000 to 76 copies, labeled 1-5) and a non-template control reaction (ntc).
  • Figure 7(a) shows realtime fluorescence signal during PCR cycling.
  • Figure 7(b) shows that the Ct value correlated tightly with the copy number of the target gene in the DNA quantification curve.
  • Figure 7(c) shows the size value (154 bp) of the product DNA as measured by post-PCR capillary electrophoresis detecting the product- incorporated TEX label . Only reactions containing template DNA resulted in a electropherogram peak, demonstrating the ability to detect specific sizes of product DNA.
  • compositions and methods are provided for gain-of-signal and target- specific detection of amplification products of polynucleotides that are easily implemented, reproducible and sensitive.
  • the gain-of-signal and target specific detection is observed after displacement of a labeled quencher or fluorescent label by polymerase-dependent extension of the polynucleotide containing the target sequence.
  • Advantages of present embodiments include at least one of the following : (a) increased sensitivity and time frame of a quantitative
  • amplification reaction (b) no additional primer optimization or probe design beyond a 5' labelled primer with a complementary detection oligonucleotide (Oligo 1/2) and unlabeled Oligo 3; (c) availability of an internal quantification standard; (d) ability to perform size detection; (e) applicability to a variety of amplification procedures; and (f) capacity for multiplexing multiple samples limited only by available fluorophors and detectors.
  • amplification reaction (b) no additional primer optimization or probe design beyond a 5' labelled primer with a complementary detection oligonucleotide (Oligo 1/2) and unlabeled Oligo 3; (c) availability of an internal quantification standard; (d) ability to perform size detection; (e) applicability to a variety of amplification procedures; and (f) capacity for multiplexing multiple samples limited only by available fluorophors and detectors.
  • the set of probes described herein for use in isothermal and PCR amplification methods utilize a sequence modified either at the 5' end or internally with either a dark quencher or a fluorophore.
  • a complementary oligonucleotide modified either at the 3' or internally with either a dark quencher or a fluorophore spectrally overlapping with the fluorophore or dark quencher of the complementary region is annealed to part of a larger single stranded polynucleotide. This creates a duplex region while leaving a single strand 3' "flap" for annealing to target nucleic acid (Oligo 2; Figure 1(a)).
  • the labels do not negatively affect the sensitivity of the amplification reaction, but the duplex region confers a delay in detection threshold time, mitigated by competitive binding between labeled quenched Oligo 1/2 and unmodified Oligo 3 ( Figure 2(a)-(d), Figure 5).
  • amplification techniques for example SDA, HDA, nicking enzyme amplification reaction (NEAR), recombinase polymerase amplification (RPA), ICAN, multiple displacement amplification (MDA), multiply primed rolling circle amplification (MPRCA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), smart amplification process (SmartAmp), ramification amplication (RAM), and genome exponential amplification reaction (GEAR), LAMP and PCR are discussed below in more detail. These examples however are not intended to be limiting.
  • the Flc regions may be 15-50 bases, for example 20-25 bases and can be designed to feature a T m from 50°C - 80°C.
  • the Oligo l :Oligo 2 duplex is optionally designed to be stably annealed at 63°C - 65°C (suitable for LAMP) and no signal is observed in the absence of strand-displacing DNA polymerase.
  • shorter primer sequences with lower melting temperature are required, for example, a specific T m is required for SNP or methylation detection, or for short regions due to weakly conserved targets, reactions can be performed at lower temperatures to accommodate less stable duplexes.
  • the Flc primer sequences provided in Table 1 range in T m from 61°C - 74°C and all perform LAMP reactions at 60°C - 65°C, showing that use of Flc: Fd duplexes does not limit primer design considerations.
  • a primer pair was also tested as described above with fluorophore and quencher positions switched on Oligo 1 (here, fluorophore) and 2 (dark quencher). Use of this reverse orientation primer set ( ⁇ ) resulted in similar amplification detection efficiency ( Figure 3(a)-(b) Figure 4(a)-(b)).
  • Oligo 3 which shares the same sequence as Oligo 1 maintains the speed and amplification detection threshold of unlabeled reactions, reducing any inhibition from duplex and labeled primers ( Figure 5).
  • primer design for PCR standard protocols in the art are used for design of the forward (F) and reverse (R) primer. This involves selecting a sequence having similar T m , and moderate G/C content.
  • the duplex region formed from Oligo 1 and Oligo 2 is designed with sufficient T m to remain annealed as duplex DNA during the amplification reaction (T m oligo 2>T A ). These parameters may be varied according to G/C content for example Oligo 2 is 48.8% G/C and has a T m of 66°C.
  • the primer may include an Oligo 2 having a length of at least 30 bases, and a T m greater than the annealing temperature and greater than or similar to the extension temperature (here 61°C and 68°C, respectively).
  • T m the annealing temperature
  • Other lengths of duplex region can be used, with the only requirement being to perform PCR reactions with extension temperatures near or below the T m of oligonucleotide 2; for example if T m of oligonucleotide is 61°C, an extension temperature of 50°C - 62°C provides sufficient annealing efficiency.
  • High affinity between Oligo 1 and Oligo 2 avoids false positives that might otherwise occur due to spurious primer annealing.
  • a fluorescent signal is observed when F2 is displaced using a probe by amplifying DNA polymerase activity.
  • the detection primer (Oligo 1) is determined by synthesis of the F and B primer with additional sequence 5' of the primer.
  • the complement of this additional sequence region (Oligo 2) is annealed to form the detection primer duplex.
  • the duplex is formed from equimolar amounts of Oligo 1 and Oligo 2 that are preformed prior to being combined with Oligo 3 in the reaction mixture.
  • fluorescence labels for use in this method includes fluorescein, 6-FAMTM (Applied Biosystems, Carlsbad, CA), TETTM (Applied Biosystems, Carlsbad, CA ), VICTM (Applied Biosystems, Carlsbad, CA ), MAX, HEXTM (Applied Biosystems, Carlsbad, CA), TYETM (ThermoFisher Scientific, Waltham, MA), TYE665, TYE705, TEX, JOE, CyTM (Amersham Biosciences, Piscataway, NJ) dyes (Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7), Texas Red ® (Molecular Probes, Inc., Eugene, OR), Texas Red-X, AlexaFluor ® (Molecular Probes, Inc., Eugene, OR)
  • BODIPY TMR BODIPY TMR
  • BOPDIPY 530/550 BODIPY 558/568
  • BODIPY 564/570 BODIPY TMR
  • Rhodamine RedTM-X Molecular Probes, Inc., Eugene, OR
  • Bright fluorophores with extinction coefficients >50,000 M “1 cm “1 and appropriate spectral matching with the fluorescence detection channels can be used to overcome loss of fluorescence signal due to dilution of template- specific Oligo 2 in multiplex reactions.
  • ROXTM Integrated DNA Technologies, Coralville, IA
  • CFX96TM Bio-Rad, Hercules, CA
  • Cy5 Cy5.5
  • HEX HEX
  • the number of different samples in a single reaction vessel and their detection is limited only by access to different fluorescent markers and to a fluorimeter with multiple channels (CFX96 as shown, 5 channels) or by the limitations of capillary electrophoresis.
  • the method described herein is amenable to high-plex amplification, such as might be achieved using fluorimeters such as the ICEPlex ® (PrimeraDx, Mansfield, MA), which can detect 60 targets using fluorescence and capillary electrophoresis, and xMAP ® (Luminex, Austin, TX), which can identify 500 targets.
  • ICEPlex ® PrimaryaDx, Mansfield, MA
  • xMAP ® Luminex, Austin, TX
  • DNA polymerase for example, Bst DNA polymerase, large fragment
  • strand displacement having an activity at elevated temperatures of, for example, 50°C - 70°C and being stable at that temperature for >30 minutes.
  • Variants of existing polymerases can be readily screened by
  • One example assay is a standard LAMP using lambda DNA (Nagamine, et al. (2002)), in which sufficient
  • amplification of 5 ng lambda phage genomic DNA is considered to be threshold detection in less than 30 minutes at 60°C - 68°C using standard primer concentrations.
  • LAMP reactions could be enhanced by using polymerase variants such as Bst 2.0 or Bst 2.0 WarmStart TM DNA
  • PCR reactions containing strand-displacing 9°N TM polymerase (New England Biolabs, Ipswich, MA) or a blend of a strand displacement polymerase and Taq (see for example, OneTaq, New England Biolabs, Ipswich, MA) provided much more robust signal than those with Taq alone, indicating without wishing to be limited by theory, that an increased efficiency of separating Oligo 2 and Oligo 1 might occur due to strand- displacement activity compared to 5'-3' exonuclease activity alone.
  • Strand- displacing DNA polymerase is included at a 0.01-0.5 ratio relative to Taq or non-strand-displacing polymerase.
  • Standard PCR polymerases that contain 5'-3' exonuclease activity (e.g. Taq DNA polymerase and variants) or strand displacement activity (e.g. 9°N, Vent " DNA polymerase (New England Biolabs, Inc., Ipswich, MA)) are suitable for use in the PCR embodiments as either activity will generate signal by separating the quencher/fluorophore duplex of Oligo 1/2.
  • 5'-3' exonuclease activity e.g. Taq DNA polymerase and variants
  • strand displacement activity e.g. 9°N, Vent " DNA polymerase (New England Biolabs, Inc., Ipswich, MA)
  • Multiplexing of samples and detection of amplification products can be achieved in a single reaction vessel as described herein.
  • a dynamic range for isothermal or PCR amplification methods for single and multiplex reactions maintains a detection sensitivity in the range of at least 5-10 9 copies of polynucleotide template with an ability to accurately detect below 1000 copies, 500 copies, 100 copies, or as few as 50 copies, 10 copies or 5 copies of a target sequence.
  • the reaction pathway for polymerase dependent extension reactions shown in Figure l(b)(i-iv) can be extended to (n) target reactions where total primer concentration may be maintained at a constant concentration, equivalent to the amount optimized in the singleplex reaction.
  • Each template-specific primer set concentration may be adjusted for the number of targets (n) in a multiplex reaction with each primer set being 1/n.
  • An internal control can be included for purposes of quantitation of the target polynucleotide (see for example, Figure 3(c)).
  • robust amplification in a multiplex environment is shown for two ( Figure 3(a)-(c)), three ( Figure 4(a)) or four (Figure 4(b)) targets.
  • amplification can maintain a level of independent performance, with each simultaneous amplification retaining sensitivity to low copy numbers and providing robust amplification.
  • This property enabled the quantitative measurement of a test target nucleic acid sample while simultaneously measuring a positive control sample, as shown for example, in Figure 3(c) for LAMP.
  • This ability to perform internal control reactions is an important diagnostic feature enabled by the described methodology.
  • Size determination can be performed by means of downstream analysis including capillary electrophoresis, which separates products based on size and can detect fluorescent labels.
  • Products from single or multiplex reactions containing fluorophore-labeled Oligo 1 can be further analyzed to determine size of product and specificity of reaction ( Figure 7(a)-(c)). This also greatly increases the degree of multiplexing, as multiple product sizes with the same fluorescent label can be distinguished by electrophoretic separation.
  • Example 1 Multiple target detection using isothermal amplification
  • Components of a LAMP reaction include:
  • Target DNA Lambda phage genomic DNA (5 ng per reaction) and HeLa genomic
  • DNA 100 ng per reaction were from New England Biolabs, Inc. (Ipswich, MA).
  • E. coil genomic DNA (5 ng per reaction) was from Affymetrix (Santa Clara, CA) and C. elegans genomic DNA (82.5 ng per reaction) was purified using standard procedures.
  • Each reaction included the following components: 1.6 ⁇ FIP (or 0.8 ⁇ FIP Oligo 3 and 0.8 ⁇ Q-FIP: Fd Oligo l : Oligo 2), 1.6 ⁇ BIP, 0.2 ⁇ F3 and B3, 0.4 ⁇ LoopF and LoopB, in addition to 0.64 U/pL Bst DNA
  • Transmembrane Conductance Regulator C R; FQ/6-FAM
  • human BRCA1 RQ/Cy5.5
  • a set of LAMP primers were adapted for bacteriophage ⁇ DNA (Nagamine, et al. (2002)) with the quencher and fluorophore positions reversed (5'-HEX Oligo 1 /3'-FQ Oligo 2) to examine any effect of quencher/fluorophore location.
  • Oligo 1/2 were made for each primer set, and LAMP reactions performed using Oligo 1/2 and Oligo 3.
  • Total oligonucleotide concentrations were kept to those described for a standard LAMP reaction (total primer concentration was kept to 5.2 ⁇ regardless of the number of templates, with each primer set adjusted by 1/n where n is number of targets in the reaction in multiplex reactions; Table 3). Reactions were performed at 65°C in triplicate, and all presented C t values represent an average ⁇ standard deviation. Results from duplex reactions are shown in Figure 3(a)-(c). Fluorescence curves from LAM P reactions result from two distinct, complete oligonucleotide sets and their corresponding genomic DNA targets. Distinct targets were detected in a single LAM P reaction. Curves shown are normalized to maximum fluorescence signal in that channel to account for differences in the signal intensity of various fluorophores.
  • the detection provided a robust signal for each target regardless of the speed of their independent amplification, which varies according to the nature of the primers, templates, and target copy number. Some amplification reactions reached exponential phase more rapidly than others. The amplification reactions with a higher Q were not affected by the faster amplification reactions in the same tube, obviating the need for consideration of amplification speed in multiplex reactions ( Figure 3(a)-(b), 4(a)-(b)).
  • Figure 3(b) (5 ng E. coli genomic DNA, ⁇ 10 6 copies) .
  • Use of the present detection method thus imposed no limitation to the sensitivity of the LAMP reaction.
  • the dynamic range of LAMP is unaffected by the detection methodology described herein in a duplex reaction, which maintained robust detection from 10-10 8 copies.
  • the detection methodology described herein can readily be extended to three and four target reactions ( Figure 4(a)-(b)), again with total primer concentration constant and each set adjusted for number of targets.
  • the primers (Table 2) contained regular PCR primers (F, Oligo 3, and R primers) at 200 ⁇ each and a pair of detection primers (Oligo 1, and Oligo 2) at 80 ⁇ each (schematic in Figure 2(c)).
  • PCR reactions were performed with Taq DNA polymerse and strand- displacing 9°N DNA polymerase using ten-fold serial dilutions of E. coli genomic DNA from 100 ng to 0.1 pg (equivalent of 20xl0 6 to 20 copies) using lx Standard Taq buffer supplemented with MgC to a final 2.25 mM, 400 ⁇ of each of the four dNTPs in 25 ⁇ reaction volume, 1.25 U Taq DNA polymerase, and 0.05U 9°N m DNA polymerase.
  • the PCR cycle and realtime signal acquisition was performed on a CFX96 machine with cycle condition at 95°C for 2 minutes; 50 cycles at 95°C for 10 seconds, 61°C for 15 seconds and 68°C for 30 seconds; final incubation at 68°C for 5 minutes.
  • the cycle number at signal that crosses the amplification threshold (Cq value, Y axis) was plotted against the log value of the copy number. As shown, the resulting qPCR data was robust and sensitive to low copy number, providing a high-R 2 (0.9969). The results are shown in Figure 6(a)-(b) .
  • C. elegans act-1 gene was detected using approximately 760,000 to 76 copies of genomic DNA in a 10-fold dilution series.
  • the act-1 reporter probe was labeled with Tex fluorescence dye and the signal was acquired using CFX96 qPCR machine.
  • the PCR cycle condition was: 95°C for 1 minute, then 50 cycles of 95°C for 10 seconds, 61°C for 15 seconds and 68°C for 30 seconds.
  • the Ct value correlated tightly with the copy number of the target gene in the DNA quantification curve ( Figure 7(a) and 7(b)).
  • the product was diluted 20- fold with water and subjected to size analysis using ABI 3130 CE instrument.
  • the expected size of the PCR product ( 154 bp) was detected in all PCR reactions containing the template DNA from 760,000 to 76 copies ( Figure 7(c)), while in the reaction containing no template DNA there was no specific peak.
  • This additional step provides further confirmation of the PCR product and thus increases the confidence of target identification.
  • both PCR and CE analysis can be automated and the combination of them would allow the high accuracy detection of large scale samples such as in patient genotyping analysis or pathogen detection.
  • LoopB ACCATCTATGACTGTACGCC (SEQ ID NO: 16)

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  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des compositions et des procédés de détection quantitative de produits d'amplification, les procédés convenant pour le multiplexage. Un premier oligonucléotide qui comprend une séquence d'amorce destinée à amorcer une réaction d'amplification et est également marqué par un marqueur fluorescent ou un extincteur est mélangé avec un deuxième oligonucléotide qui présente une séquence appropriée pour l'hybridation d'une partie du premier oligonucléotide et présente un marqueur fluorescent si le premier oligonucléotide présente un extincteur ou un extincteur si le premier oligonucléotide présente un marqueur fluorescent ; et un troisième nucléotide qui comprend une partie ou toute la séquence d'amorce contenue dans le premier oligonucléotide mais n'est pas marqué, le premier et le troisième oligonucléotide étant combinés dans un rapport molaire de 2,8 à 8,2.
PCT/US2013/052189 2012-07-27 2013-07-26 Détection de produits d'amplification WO2014018828A1 (fr)

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US10253357B2 (en) 2014-04-24 2019-04-09 Diassess Inc. Colorimetric detection of nucleic acid amplification
US11584957B2 (en) 2014-04-24 2023-02-21 Lucira Health, Inc. Colorimetric detection of nucleic acid amplification
US11123736B2 (en) 2016-03-14 2021-09-21 Lucira Health, Inc. Systems and methods for performing biological assays
US11291995B2 (en) 2016-03-14 2022-04-05 Lucira Health, Inc. Selectively vented biological assay devices and associated methods
US12090482B2 (en) 2016-03-14 2024-09-17 Pfizer Inc. Systems and methods for performing biological assays
US12023671B2 (en) 2016-03-14 2024-07-02 Pfizer Inc. Selectively vented biological assay devices and associated methods
US11125661B2 (en) 2016-03-14 2021-09-21 Lucira Health. Inc. Devices and methods for biological assay sample preparation and delivery
US12023665B2 (en) 2016-03-14 2024-07-02 Pfizer Inc. Devices and methods for modifying optical properties
CN110536968A (zh) * 2016-12-23 2019-12-03 阿尔伯特-路德维希-弗莱堡大学 两部分式中介探针
KR20190135468A (ko) * 2016-12-23 2019-12-06 알베르트-루드빅스-유니베르지텟 푸라이부르그 2-조각 매개체 프로브
KR102523355B1 (ko) 2016-12-23 2023-04-20 알베르트-루드빅스-유니베르지텟 푸라이부르그 2-조각 매개체 프로브
WO2018114674A1 (fr) * 2016-12-23 2018-06-28 Albert-Ludwigs-Universität Freiburg Sonde médiatrice en deux parties
US11954851B2 (en) 2017-04-06 2024-04-09 Pfizer Inc. Image-based disease diagnostics using a mobile device
US11080848B2 (en) 2017-04-06 2021-08-03 Lucira Health, Inc. Image-based disease diagnostics using a mobile device
US11465142B2 (en) 2017-09-14 2022-10-11 Lucira Health, Inc. Multiplexed biological assay device with electronic readout
USD910200S1 (en) 2018-12-21 2021-02-09 Lucira Health, Inc. Test tube
USD953561S1 (en) 2020-05-05 2022-05-31 Lucira Health, Inc. Diagnostic device with LED display
USD962470S1 (en) 2020-06-03 2022-08-30 Lucira Health, Inc. Assay device with LCD display

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