WO2015023616A2 - Procédé de discrimination exhaustive, quantitative et hautement sensible de séquences d'acides nucléiques dans des populations homogènes et hétérogènes - Google Patents

Procédé de discrimination exhaustive, quantitative et hautement sensible de séquences d'acides nucléiques dans des populations homogènes et hétérogènes Download PDF

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WO2015023616A2
WO2015023616A2 PCT/US2014/050613 US2014050613W WO2015023616A2 WO 2015023616 A2 WO2015023616 A2 WO 2015023616A2 US 2014050613 W US2014050613 W US 2014050613W WO 2015023616 A2 WO2015023616 A2 WO 2015023616A2
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sequences
melt curves
target sequence
curves
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WO2015023616A3 (fr
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Samuel Yang
Jeff Wang
Stephanie I. FRALEY
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Johns Hopkins University
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • This invention relates to the field of biotechnology and, more specifically, to methods of measuring nucleic acids in homogeneous and heterogeneous samples.
  • MicroRNA profiling approaches and considerations. Nature Reviews Genetics, 13:358-369 (2012); Blainey, P.C. The future is now: single-cell genomics of bacteria and archaea. FEMS Microbiol Rev (2013); Yang, S. and Rothman, R.E. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis, 4:337-348 (2004).
  • hybridization assays such as microarrays are used for broad, semi-quantitative profiling, while multi-reaction quantitative PCR (qPCR) is used for enumeration of multiple species.
  • the qPCR arrays currently available require that the sample input be divided among multiple wells resulting in multiple target molecules per well and only one primer set per well targeting an individual species as shown in Figure 1(b); sensitivity is inversely related to the number of reactions. Low level species may not be distributed to a well containing appropriate primers, and unknown species will not be discovered.
  • Multiplexed qPCR involves a single reaction containing all target molecules and multiple sets of specific primers and probes targeting individual species; single copy sensitivity is possible as shown in Figure 1(c).
  • the number of detectable target species is limited by the resolution of fluorescent probe spectra to about 4 species and unknown targets will not be discovered.
  • Microarrays and qPCR techniques lack the sensitivity to detect species in low level concentrations since the sample must be split across many reactions containing distinct sets of primers or probes.
  • NGS next generation sequencing
  • RT-PCR Real Time - PCR
  • bulk HRMA has previously been described in WO2009/134470 (US2013/0217588A1), which is incorporated herein by reference in its entirety.
  • a method for the detection of nucleic acid sequences in a sample is provided.
  • the first step of the method is to conduct an optimized digital PCR (dPCR) reaction of the sample to amplify the target sequences.
  • dPCR digital PCR
  • a HRMA is conducted to obtain the melt curves of the dPCR amplified product.
  • the melt curves of the sample are compared to the standard melt curves of the target sequence to identify the particular sequences found in the sample.
  • a method for the detection of nucleic acid sequences in a heterogeneous sample comprises: amplifying at least one target sequence individually in a sample through a digital PCR (dPCR) procedure, obtaining the individual melt curves of the at least one individually amplified target sequence in the sample through dHMRA, wherein the dHRMA procedure allows resolution of individual melt curves for each target sequence as opposed to an average melt curve for the sample, and comparing the melt curves in the sample to a set of standard melt curves for the target sequence.
  • the sequences are amplified through broad-based primers or universally ligated adapter sequences.
  • the sample contains at least two target sequences.
  • the dPCR procedure comprises amplification by digitizing the sample to have a single molecule per reaction well.
  • the digitizing step comprises diluting the sample to achieve a digital dilution within a digital range of one or two out of three reactions providing a negative result.
  • a method for identifying infectious pathogens comprises individually amplifying at least one target sequence in a sample containing a heterogeneous mixture of pathogen DNA, wherein said amplification is conducted through dPCR, obtaining the individual melt curves of the at least one target sequence in the sample through dHMRA, wherein the dHRMA procedure allows resolution of individual melt curves for each target sequence as opposed to an average melt curve for the sample, and comparing the melt curves in the sample to a set of standard melt curves for target sequences, wherein the target sequence is a unique sequence of one infectious pathogen.
  • the method can be used to identify one or more pathogens selected from the group consisting of S. aureus, S. epidermidis, S. saprophyticus, S.
  • the sequences are amplified through broad-based primers or universally ligated adapter sequences.
  • a method for miR A profiling comprises individually amplifying at least one target sequence in a sample containing a heterogeneous mixture of miRNAs, wherein said amplification is conducted through optimized dPCR reactions that enhance specificity of amplification, obtaining the individual melt curves of the at least one target sequence in the sample through dHMRA, wherein the dHRMA procedure allows resolution of individual melt curves for each target sequence as opposed to an average melt curve for the sample, and comparing the melt curves in the sample to a set of standard melt curves for target sequences, wherein the target sequence is a unique sequence of one miRNA.
  • Figure 1 (a) shows a graphical representation of digital High Resolution Melt Analysis (dHRMA).
  • Figure 1 (b) shows a graphical representation of qPCR assays.
  • Figure 1 (c) shows a graphical representation of multiplex qPCR assays.
  • Figure 1 (d) shows a graphical representation of qPCR assays followed by bulk HRMA.
  • Figure 2 (a) is a graphical representation of the raw HRM data showing the loss of fluorescence in wells containing standard dilutions of four tagged Let-7 miRNA sequences (unmixed).
  • Figure 2 (b) is a derivative plot of raw fluorescence data from two separate experiments containing 4 dilutions each that has been temperature shifted by alignment of the temperature calibrator curve peaks, vertical dotted lines.
  • Figure 2 (c) is graphical representation of multi-experiment data from Figure 2(b) after normalization and temperature calibration showing highly reproducible, unique melting curves for each tagged miR A sequence and no template controls are clearly distinguishable (gray curves).
  • Figure 2 (d) shows normalized, temperature calibrated, standard curves from two independent dilution series experiments using each Let-7 family and related miRNA sequence give database references for future dHRMA target identification.
  • Figure 3 (a) is a graphical representation of the raw HRM data showing the loss of fluorescence in each well of a 96-well plate across which a dilute mixture of Let-7a, b, c, and miR-29 was dispersed.
  • Figure 3 (b) shows normalized, temperature calibrated, and database matched melt curves show universally amplified, single copy detection of each of the four input miRNA (colored melt curves), detection of wells containing multiple copies of targets (light gray, non-matching curves), and amplification negative wells (dark gray melt curves). The number of copies detected is shown in parentheses next to each legend label.
  • Figure 3 (d) shows standard database curves used to identify target melt curves.
  • Figure 4 (a) shows normalized and temperature calibrated database melt curves for each of the clinically relevant bacteria listed in Table 2 are resolvable, demonstrating the high sensitivity of HRMA.
  • Figure 4 (b) shows difference curves of each bacteria strain using S. aureus as a reference. In previously work which didn't include temperature calibrators or PCR optimal buffers, V6 amplicons of S. aureus, S. epidermidis, and S. saprophyticus were not resolvable by HRMA (14).
  • Figure 5 (a and b) show derivative plots of HRM fluorescence data before temperature shifting; melting curves for the same species do not accurately overlap. Inset in a is an enlarged view of the low temperature calibrator melt curve showing slight differences in melting peak.
  • Figure 5 (c and d) show derivative plots of HRM fluorescence data after temperature shifting showing improved matching and resolution. Four 10-fold dilutions of each of the four species are plotted.
  • Figure 6 (a) shows raw dHRMA data showing the loss of fluorescence in all but one well of a 96-well plate across which two dilute mixtures of S. aureus, E. faecalis, and P. acnes were dispersed in two separate experiments.
  • Figure 6 (b) shows normalized, temperature calibrated, and database matched digital melt curves after 70 cycles of PCR show broad-based amplified, single copy detection of each of the four input bacteria (colored melt curves), detection of wells containing multiple copies of input or contaminating gDNA templates from Taq polymerase (light gray, non-matching curves), and amplification negative wells (dark gray melt curves). The number of copies detected is shown in parentheses next to each legend label.
  • Figure 7 shows uMELT model predictions of 100 distinct bacterial amplicon melt curves demonstrating the potential for resolving numerous organisms by dHRMA.
  • dHRMA Digital high resolution melt analysis
  • Nucleic acid molecules of interest are partitioned, as in digital PCR, and amplified using broad-based primers or universally ligated adapter sequences.
  • HRMA specific DNA intercalating dye.
  • Extensive optimization of the assay enables a database of known target melt curves to be used for identification. Absolute quantitation of numerous and even unknown genotypes with single molecule and single nucleotide sensitivity is possible in a single assay.
  • the present invention further provides for the use of dPCR followed by HRMA to isolate and identify target sequences from heterogeneous mixtures.
  • the combination of dPCR and HRMA provides a digitized HRMA method, or dHRMA, in which the melt curves of each individual target sequence can be determined.
  • One method of the present invention consists of the following steps. In a first step, the sample is "digitized" via dilutional or microfluidic methods. Digitization partitions target DNA/R A species into individual single molecule samples. In a second step, the samples are amplified through universal amplification as described in more detail below. In a third step, HRMA of the digitally amplified product is conducted providing individual melt curves for each partitioned molecule. Finally, the melt curves for each individually partitioned molecule are compared to a database of melt curves for identification of the target sequence.
  • Digitization enables HRMA to accomplish both identification and quantification of many more target molecules than traditional HRMA methods ( Figure 1).
  • dHRMA uses a diluted sample input such that either zero or one copy of the target molecule is distributed per reaction well. It is contemplated that other methods may be used to achieve digitation. For example, microfluidic technologies may be used that partition samples into minute reaction volumes, each holding a single target molecule of interest. Broad-based primers amplify all targets individually giving a singular melt curve for each target/reaction and allowing quantification and identification of all species, including unknown or unexpected species, in the mixture.
  • a "digital level" of amplification is reached by serial dilutions, where each dilution is run through PCR and HRM in triplicate.
  • the digital dilution is identified as the particular dilution that after PCR and HRM gives some positive and some negative signals out of the three replicate reactions.
  • Each tenfold dilution is run in triplicate, and the first dilution where one or two out of three reactions is negative is considered in the digital range.
  • the level of digitization is determined by quantifying the number of positive and negative reactions and fitting those numbers to a Poisson distribution.
  • dHRMA is an extension of dPCR
  • the same principles apply: the distribution of digital melt curves is governed by stochasticity and the quantification of species by summation of dHRMA melt curve types can achieve greater precision than qPCR quantification.
  • the number of each type of melt curve relates to the original concentration of individual species in the heterogeneous sample by Poisson statistics.
  • the integration of three key techniques enable the multiplexing and accuracy achieved by the dHRMA assay: (1) the incorporation of broad- based primers or universally ligated priming sites for unbiased amplification of all molecules of interest; (2) shifting the burden of discriminating all amplified sequences from primers and probes to digital melt curves for specificity that relies on the inherent physical properties of the sequence flanked by conserved primer sequences; (3) highly optimized reaction conditions that incorporate tools for normalization to enable database matching.
  • dHRMA melt curves relies first on optimal dPCR reaction conditions such that single copies of template are reliably amplified, primer-dimers and non-specific amplification products are averted, and the reaction is cycled to completion. This precludes false negative or erroneous melt curves in the downstream analysis. Thus primer concentration is minimized and cycling extended during optimization. Primer specificity is also important since dHRMA is highly sensitive to even single nucleotide differences in amplicon sequence, but relying on annealing temperature for control of specificity is risky due to the inherent technical challenges of ensuring uniform heating across all reactions and the need to adapt for each primer set involved.
  • buffer conditions may be optimized by including ammonium and potassium ions which universally stabilize specific annealing and destabilize non-specific hydrogen bonding respectively.
  • the final concentrations are the following: Potassium or ammonium salt at 50mM, betaine at 500mM, 10% poly(ethylene glycol) by weight, universal primers at 400nM, lOnM fluorescein, 3.5mM MgC12, 50nM temperature calibrators, IX Evagreen dye, 0.2mM dNTP, 0.05U/ul Taq polymerase. This promotes specificity across a wide range of annealing temperatures for any primers. Using universal and broad-based primers ensures that primer specificity is equivalent across all targets so that discrimination of species relies only on the sequence between the conserved priming sites.
  • PCR reagents often contain background levels of bacterial genomic DNA (gDNA), particularly Taq polymerases generated with recombinant DNA in bacterial cultures. Indeed, approximately 1-1.5 copies of contaminating gDNA per well on average was observed in initial dHRMA experiments. This level of background in the PCR reagents obscured digitization of the target sequences and resulted in complex multi-species melting curves within the majority of the wells. Contamination is reduced using a filtration protocol, as recognized by a person of ordinary skill in the art.
  • all PCR reagents except Taq polymerase and target gDNA are first mixed and then filtered to remove contaminating microbial DNA.
  • a highly purified Taq polymerase with very low background DNA contamination is preferably used.
  • All water used for polymicrobial experiments is treated with DNase and then heat inactivated prior to use.
  • reduction in reaction volume assists in overcoming contamination problems and facilitates digitization. Reaction volumes are reduced in half, which, in turn, reduces the average level of contamination by half but maintains the same reagent concentrations.
  • Universal amplification means the co-amplification of a heterogeneous nucleic acid sequence population of interest (e.g., polyallelic genetic loci, small or large RNA species, etc.). Universal amplification is accomplished through the use of universal tag sequences.
  • the universal tag sequences in one embodiment, are primers designed against highly conserved sequences flanking hypervariable regions of the target DNA/RNA. The tag sequences are screened for homology with other non-target sequences and tested for their propensity to form hairpin structures. Structures with low propensity to form such structures are preferred as they would provide a more efficient replication reaction.
  • polymicrobial pathogen primers are designed based on common sequences among various pathogens.
  • universal tag sequences consist of primers that include ligation-mediated adaptors or multiplexing primers that include built-in label sequences for universal priming.
  • multiplex ligation dependent probe amplification also known in the art as MPLA
  • Ligation-mediated adaptors are small oligonucleotides designed bind to target nucleic acid fragments of varying sequences but which only undergo replication if the complementary adaptors are present.
  • PCR primers which anneal to the built-in label sequences on the adaptors, are then used to co-amplify all the target fragments.
  • multiplexing primers e.g. molecular inversion probes
  • designed to capture multiple target sequences of interest have built-in label sequences to allow universal amplification of all captured sequences.
  • the universal tag sequences are utilized to optimize the reactions for dHRMA. Each universal tag sequence is used in known samples to develop standard melt curves and a database of standard melt curves is assembled. The universal tag sequence standard melt curves or target sequence curves are used to assess the presence of the sequence of interest in a heterogeneous sample in the final step of the method described above.
  • a genomic DNA sample is subjected to the optimized dHRMA reactions for the particular target sequences.
  • the gDNA sample, or any target sample suspected to have the target sequences is subjected to dPCR in accordance with the optimal conditions for the dHRMA reactions.
  • the sample is first digitized and then subjected to PCR utilizing the target sequence primers.
  • the dPCR reaction amplifies the sequences found in the heterogeneous sample.
  • the dPCR reactions are subjected to HRMA generating individual melt curves per sample.
  • the melt curves for the sample are identified utilizing standard scanning techniques.
  • DNA intercalating agents are added to the solution in some preferred embodiments.
  • the generated melt curves are compared to the standard melt curves in order to identify the amplified sequences.
  • the dHRMA method described allows highly sensitive, specific, broad-based detection and discovery of potentially thousands of nucleic acid genotypes in a rapid single assay format that is less expensive and generally more accessible than NGS.
  • This new technique unites and Stahls aspects of limiting dilution/ digital PCR (dPCR) (Vogelstein, B. and Kinzler, K.W. Digital PCR. Proc. Natl. Acad. Sci., 96:9236-9241(1999); Sykes, P.J., et al. Quantitation of Targets for PCR by Use of Limiting Dilution. Biotechniques, 13:444- 449 (1992)), broad-based/universal amplification (Yang, S., et al.
  • dHRMA for pathogen detection overcomes sensitivity and specificity issues resulting from contamination by environmental microbes, contamination within PCR reagents, and multi-species polymicrobial infections, which can be problematic to diagnostic assays (Corless, C.E., et al. Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR. Journal of Clinical Microbiology, 38: 1747-1752 (2000); Garcia, P. Coagulase-negative staphylococci: clinical, microbiological and molecular features to predict true bacteraemia. Journal of Medical Microbiology, 53:67-72 (2004); Weinstein, M.P., et al.
  • HRMA is performed using DNA saturating dyes or multiple color probes to generate a melt signature that uniquely identifies each specific bacterial species. Even though a generally reproducible melting signature was obtained when each of the > 50 bacterial species was measured independently, the species could not be individually identified when multiple species were present in the sample simultaneously or when contaminating bacteria were present, as often occurs clinically.
  • dHRMA can be used for profiling circulating miRNA in clinical samples and monitoring host immune response. miRNA are short (19-22nts) non-coding RNA that interact with messenger RNA to regulate gene expression. Circulating miRNA, released by cells into the blood are ideal candidates for quantitative profiling by the highly sensitive dHRMA assay. Quantitation, despite their low level presence, is possible via digitization, and their short lengths give greater melt curve shifts among different sequences for accurate resolution of single nucleotide differences.
  • dHRMA absolute quantification and identification of numerous target genotypes, including discovery of unexpected or unknown species, in a heterogeneous sample using a generic reporter dye, such as EvaGreen (Sigma), is possible by dHRMA.
  • This technique uses limiting dilution digitization to partition target nucleic acids across many reactions allowing discretized HRMA, where each sample molecule is represented by a single and specific melting profile.
  • Broad-based primers or universal tagging allows unbiased amplification of all nucleic acids of interest.
  • Sample-to-answer is achieved with a single assay in a few hours, circumventing the costly, lengthy, and multi-step process of NGS.
  • Single nucleotide resolution, single molecule sensitivity, and broad-based multiplexing offer improvements to traditional microarray and qPCR profiling.
  • the dHRMA approach is used for sensitive, quantitative, and accurate detection in mock heterogeneous samples containing multiple pathogens and contaminants commonly involved in polymicrobial sepsis (2) or members of the Lethal-7 (Let-7) family of closely related host miRNA known to be key infection-related biomarkers (Schulte, L.N., et al. Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family. EMBO J, 30: 1977-1989 (2011)). This technology can identify many more microbes or miRNAs. Strategies to further expand the number of targets identifiable by dHRMA include incorporating multiple broad-based primer sets and unlabeled probes or generating longer amplicons (Figure 7).
  • the dHRMA curve matching method allows distinction of single sequences in polymicrobial and miR A experiments ( Figures 3B and 6B).
  • absolute quantities of each sequence are resolved by expanding the number of reactions across which the sample is diluted. More reactions assist in ensuring that an entire sample can be processed, that individual species are identified separately, and that the likelihood of two or more sequences occupying the same well is extremely low (Table 3).
  • the precision of digital quantification is also improved by increasing the number of reactions Dube, S., et al. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device. PLOS ONE, 3 (2008).
  • the ability to quantify the heterogeneity of species present in a sample may aid clinicians in identifying true sepsis versus contamination in an otherwise ambiguous sample where some bacteria can function in either category, e.g. coagulase negative staphylococci.
  • Biomarker research may also significantly benefit from this technology.
  • miRNA profiling the need for highly specific, quantitative, and all-encompassing profiling that is quickly and easily obtained has not been met by any singular technology. The ability of a single platform to accomplish all of these tasks increases the interpretability and reproducibility of findings in the field of nucleic acid identification.
  • a method for building a dHRMA curve standard library is provided.
  • a standard library of melt curves is developed through the digitization process described herein.
  • the melt curves of known sequences are identified and stored for use as a reference library.
  • a mixture of known target molecules is prepared for dHRMA by diluting the sample to achieve a single molecule per reaction configuration in a multiwell plate.
  • the known targets are then amplified utilizing dPCR.
  • the amplified targets are then subject to HRMA and the melt curves are identified.
  • the melt curve standard library identified can then be utilized to evaluate heterogeneous samples containing unknown mixtures of DNA.
  • S. aureus, E. faecalis, and P. acnes were identified at the single cell level by dHRMA using broad-based 16s rRNA primers.
  • a database of the Lethal-7 (Let-7) family and two related microRNA was also generated.
  • Single copies of Let-7a, Let- 7b, Let- 7c, and miR-29 were identified by dHRMA using universal primers.
  • Tag-F (5'- CCATAGACGTAGCAACGATCG-3 ')(SEQ ID No. 1)
  • Tag-R 5 ' -GATGC AAGG ACT ATCCACTCAC-3')(SEQ ID No. 2).
  • tagged cDNA corresponding to the ten miRNA sequences in Table 1 were synthesized by Integrated DNA technologies (IDT, Coralville, IA).
  • V6-F 5'- GGAGCATGTGGTTTAATTCGA-3 '
  • V6-R 5 '-AGCTGACGACANC CATGCA-3'
  • DNA was extracted from clinically isolated or American Type Culture collection (ATCC) acquired bacterial strains: ⁇ , lugdunensis, S. enteritidis, S. aureus, S. choleraesuis, S. saprophyticus, S. epidermidis, S. Dublin, K. pneumoniae, E. faecalis, P. acnes, P. aeruginosa, S. agalactiae) using a Roche MagNA Pure LC (Roche Diagnostics, Indianapolis, IN) with the DNA Isolation Kit I (Roche Diagnostics) using a 200 ⁇ input volume and a 100 ⁇ final elution volume per manufacturer's instructions.
  • ATCC American Type Culture collection
  • Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons.
  • White 96-well plates with black semi-skirting were used to maximize detection of fluorescence signal and minimize well-to-well fluorescence cross-talk (Eppendorf).
  • Optimized dHRMA miRNA reactions were performed as follows: lOul total reaction volume consisting of IX PCR buffer (Qiagen), lOnM fluorecene (Bio-rad), 3.5mM total MgC12 (Qiagen), 400nM of each tag primer (IDT), 50nM temperature calibrator sequences (IDT), IX EvaGreen (Sigma), 200uM dNTP (Invitrogen), 0.05U/ul HotStart Taq polymerase (Qiagen), 2ul gDNA dilution, and ultrapure water (Quality Biological), with a 15ul overlay of high quality DNase-free mineral oil (Sigma).
  • Thermocycling proceeded as follows: hold- (95°C 5min), cycle 60 times-(95°C 30sec, 59°C 30sec), and cycle 1 time-(95°C 30sec, 25°C hold).
  • hold- 95°C 5min
  • cycle 60 times- 95°C 30sec
  • 59°C 30sec 59°C 30sec
  • Single copy amplification by tag primers clustered around a PCR cycle threshold (Ct) of 45.
  • thermocycling conditions that achieved reliable single copy amplification near a Ct of 45 were chosen. To ensure amplification completion, 70 cycles were run. The optimized thermocycling conditions were: hold-(95°C 5min), cycle 70 times-(95°C 30sec, 65°C 30sec, 72°C 30sec), and cycle 1 time- (95°C 30sec, 25°C hold).
  • dHRMA was performed on the 96-well digital plate with LightScanner equipment (BioFire Diagnostics, Salt Lake City, UT) using a temperature range from 55 to 95°C. Analysis was accomplished using the LightScanner software with Call-IT 2.5 small amplicon genotyping algorithm, which incorporates temperature shifting and normalization using the low and high temperature calibrators. The multi-plate analysis tool was used to match standard database curves to experimental curves and identify the templates.
  • FIG. 2A shows the raw fluorescence melt data for Let-7a, Let- 7b, Let-7c, and miR-29. A derivative plot of the fluorescence data was generated and alignment and normalization according to the low and high temperature calibrator sequences was performed ( Figure 2B).
  • Figure 2 C shows the calibrated and normalized melt data as a percentage of the highest fluorescence, i.e. when amplicons are fully annealed in a helical structure.
  • Wells negative for amplification are easily distinguishable (gray lines, Figure 2C).
  • Optimization of reaction conditions resulted in highly reproducible melt curves for each sequence in Table 1, and these were readily distinguishable using the LightScanner's small amplicon genotyping algorithm (Figure 2D).
  • the melting curves in Figure 2 were collected over multiple dilutions and multiple days of experimentation, demonstrating the reliability of the optimized assay.
  • FIG. 3 shows the results of universal dHRMA for the mixture of miRNA. Normalized melt curves were reliably matched to a previously generated database of temperature calibrated melt curves for each miRNA (Figure 3B). From the mixture, 14 Let-7a miRNA were detected, along with 12 Let- 7b, 2 Let-7c, and 8 miR-29 sequences. Of 96 reactions, 11 were unidentified, meaning the melt curve resulted from a combination of two or more of the input sequences and 49 reactions were negative.
  • melt curves represent single copies of unknown gDNA templates, though not necessarily all distinct from one another. These may have originated either from the PCR reagents themselves or potentially from very low level contaminants amplified by culture and carried over into the spiked gDNA input.
  • Estimates of Taq contamination as stated by the manufacturer are ⁇ 10 copies/ul Taq, which gives a ⁇ PGR contaminants ⁇ 0.5 for current polymicrobial dHRMA assay.
  • the original input mixture of S. aureus, E. faecalis, and P. acnes gDNA then has ⁇ i nput ⁇ 0.44. With an increased number of digital reactions, absolute quantitation of each bacterial species can be achieved.
  • Table 1 Shows the differences in nucleotide sequences for the Lethal-7 family and related miRNA. The differences are underlined.
  • let-7f TGAGGTAGTAG ⁇ TTGTATAGTT 1 nt 12
  • let-7g TGAGGTAGTAG7TTGTACAGT 2 nt 13
  • Table 2 provides a list of clinically relevant bacteria involved in sepsis.
  • Table 3 shows the dynamic range of dHRMA detection as a function of the number of reactions.
  • the present invention is applicable to methods for identification of biological molecules.
  • the invention discloses a method for the detection of nucleic acids in heterogeneous samples.
  • the method and devices described herein can be made and practiced in industry in the field of biotechnology.

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Abstract

La présente invention concerne un procédé de détection d'acides nucléiques dans des échantillons hétérogènes. De très petites quantités d'acides nucléiques peuvent être identifiées dans un échantillon par dHRMA (Analyse numérique à haute résolution des courbes de fusion). Lors d'une étape du procédé, on identifie des courbes de fusion pour des séquences cibles spécifiques. Lors d'une deuxième étape, des amorces d'amplification des séquences cibles sont utilisées dans le cadre d'une PCR numérique optimisée en vue de la détection des séquences cibles. Lors d'une étape ultérieure, les courbes de fusion des séquences présentes dans l'échantillon sont obtenues. Lors de la dernière étape, les courbes de fusion obtenues à partir de l'échantillon sont comparées aux courbes de fusion étalons pour les séquences cibles.
PCT/US2014/050613 2013-08-12 2014-08-12 Procédé de discrimination exhaustive, quantitative et hautement sensible de séquences d'acides nucléiques dans des populations homogènes et hétérogènes Ceased WO2015023616A2 (fr)

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CN104894283A (zh) * 2015-06-25 2015-09-09 蔡先全 检测沙门氏菌及整合子int的引物、试剂盒及方法
WO2018094091A1 (fr) 2016-11-17 2018-05-24 Combinati Incorporated Procédés et systèmes d'analyse et de quantification d'acide nucléique
WO2018119443A1 (fr) * 2016-12-23 2018-06-28 The Regents Of The University Of California Procédé et dispositif de fusion numérique haute résolution
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WO2023163664A3 (fr) * 2022-02-28 2023-10-26 National University Of Singapore Procédé de profilage d'une composition microbiotique
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US11285478B2 (en) 2016-04-04 2022-03-29 Combinati Incorporated Microfluidic siphoning array for nucleic acid quantification
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US11697844B2 (en) 2016-11-17 2023-07-11 Combinati Incorporated Methods and systems for nucleic acid analysis and quantification
JP2024028879A (ja) * 2016-11-17 2024-03-05 コンビナティ インコーポレイテッド 核酸分析および定量化のための方法ならびにシステム
JP2021118692A (ja) * 2016-11-17 2021-08-12 コンビナティ インコーポレイテッド 核酸分析および定量化のための方法ならびにシステム
EP3541519A4 (fr) * 2016-11-17 2020-07-01 Combinati Incorporated Procédés et systèmes d'analyse et de quantification d'acide nucléique
EP3984642A1 (fr) * 2016-11-17 2022-04-20 Combinati Incorporated Procédés et systèmes d'analyse et de quantification d'acide nucléique
US12091711B2 (en) 2016-11-17 2024-09-17 Combinati Incorporated Methods and systems for nucleic acid analysis and quantification
WO2018094091A1 (fr) 2016-11-17 2018-05-24 Combinati Incorporated Procédés et systèmes d'analyse et de quantification d'acide nucléique
JP7490610B2 (ja) 2016-11-17 2024-05-27 コンビナティ インコーポレイテッド 核酸分析および定量化のための方法ならびにシステム
JP2019535247A (ja) * 2016-11-17 2019-12-12 コンビナティ インコーポレイテッド 核酸分析および定量化のための方法ならびにシステム
EP4617381A3 (fr) * 2016-11-17 2025-11-05 Combinati Incorporated Procédés et systèmes d'analyse et de quantification d'acide nucléique
EP4282981A3 (fr) * 2016-11-17 2024-01-17 Combinati Incorporated Procédés et systèmes d'analyse et de quantification d'acide nucléique
JP7709505B2 (ja) 2016-11-17 2025-07-16 コンビナティ インコーポレイテッド 核酸分析および定量化のための方法ならびにシステム
WO2018119443A1 (fr) * 2016-12-23 2018-06-28 The Regents Of The University Of California Procédé et dispositif de fusion numérique haute résolution
US11915795B2 (en) 2016-12-23 2024-02-27 The Regents Of The University Of California Method and device for digital high resolution melt
EP3559261A4 (fr) * 2016-12-23 2020-12-23 The Regents of The University of California Procédé et dispositif de fusion numérique haute résolution
US11390902B2 (en) 2018-01-22 2022-07-19 Luminex Corporation Methods and compositions for discrete melt analysis
US12465915B2 (en) 2018-12-10 2025-11-11 Combinati Incorporated Microfluidic array for sample digitization
US12330151B2 (en) 2020-09-28 2025-06-17 Combinati Incorporated Devices and methods for sample processing
WO2023163664A3 (fr) * 2022-02-28 2023-10-26 National University Of Singapore Procédé de profilage d'une composition microbiotique
CN115029462B (zh) * 2022-08-10 2022-11-08 中国农业科学院农业质量标准与检测技术研究所 一种动植物疫病的核酸标准物质、形貌模拟方法及其应用
CN115029462A (zh) * 2022-08-10 2022-09-09 中国农业科学院农业质量标准与检测技术研究所 一种动植物疫病的核酸标准物质、形貌模拟方法及其应用

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