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Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

Fraley SI, Hardick J, Jo Masek B, Athamanolap P, Rothman RE, Gaydos CA, Carroll KC, Wakefield T, Wang TH, Yang S - Nucleic Acids Res. (2013)

Bottom Line: The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR.We show that single-molecule detection and single nucleotide sensitivity is possible.U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA, Department of Emergency Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Infectious Disease, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University, Baltimore, MD 21218, USA, The Johns Hopkins Hospital, Baltimore, MD 21287, USA and Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

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Twelve clinically relevant bacteria are resolvable by HRM. (a) 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 HRM. (b) Difference curves of each bacteria using S. aureus as a reference. In previously work that did not include temperature calibrators or PCR optimal buffers, V6 amplicons of S. aureus, S. epidermidis and S. saprophyticus were not resolvable by HRM (14).
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gkt684-F4: Twelve clinically relevant bacteria are resolvable by HRM. (a) 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 HRM. (b) Difference curves of each bacteria using S. aureus as a reference. In previously work that did not include temperature calibrators or PCR optimal buffers, V6 amplicons of S. aureus, S. epidermidis and S. saprophyticus were not resolvable by HRM (14).

Mentions: Here, we demonstrate that U-dHRM with broad-based bacterial 16s rDNA primers targeting the hypervariable V6 region and DNA intercalating dye can resolve each bacteria within a mixture of pathogens involved in polymicrobial sepsis and common clinical contaminants (Table 2). Melt curves were generated from standard dilutions reaching the digital level to ensure accurate curves for database creation. Calibrated and normalized helicity and difference curves of 12 clinically relevant bacteria were experimentally generated for the database (Figure 4). Highly pure and concentrated gDNA from laboratory stock organisms was used to generate standard melt curves, as any cross-contamination by other bacterial DNA would be amplified by the universal primers and contribute to changes in the melt curves. The amplicons were subsequently sequenced for identity validation (Supplementary Table 1). Difference curves of S. aureus, S. epidermidis and S. saprophyticus V6 amplicons were previously unresolvable (14), but under the currently optimized U-dHRM conditions are highly reproducible and discernible using only intercalating dye as the reporter (Figure 5). Next, a spiked mixture of S. aureus, E. faecalis and P. acnes was prepared, digitized, amplified by V6 broad-based primers and assessed by U-dHRM. Figure 6 contrasts a bulk HRMA experiment (red melt curve) and the U-dHRM results (all other melt curves) of the polymicrobial mixture. Input concentrations were adjusted such that the same amount of the polymicrobial gDNA was added to the bulk well as was distributed across the digital wells. Four curves were positively identified by matching to the database curves (Figure 6D). These were amplified from singular target templates. Knowing the number of negatives, 37, and total positives, 58, a Poisson calculation gives λoverall = 0.94 for the reaction mixture (Figure 6C). By this calculation, ∼31 of the unidentified 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 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/µl Taq, which gives a λPCR contaminants ∼ 0.5 for the polymicrobial U-dHRM assay. The original input mixture of S. aureus, E. faecalis and P. acnes gDNA then has λinput ∼ 0.44.Figure 4.


Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.

Fraley SI, Hardick J, Jo Masek B, Athamanolap P, Rothman RE, Gaydos CA, Carroll KC, Wakefield T, Wang TH, Yang S - Nucleic Acids Res. (2013)

Twelve clinically relevant bacteria are resolvable by HRM. (a) 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 HRM. (b) Difference curves of each bacteria using S. aureus as a reference. In previously work that did not include temperature calibrators or PCR optimal buffers, V6 amplicons of S. aureus, S. epidermidis and S. saprophyticus were not resolvable by HRM (14).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794612&req=5

gkt684-F4: Twelve clinically relevant bacteria are resolvable by HRM. (a) 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 HRM. (b) Difference curves of each bacteria using S. aureus as a reference. In previously work that did not include temperature calibrators or PCR optimal buffers, V6 amplicons of S. aureus, S. epidermidis and S. saprophyticus were not resolvable by HRM (14).
Mentions: Here, we demonstrate that U-dHRM with broad-based bacterial 16s rDNA primers targeting the hypervariable V6 region and DNA intercalating dye can resolve each bacteria within a mixture of pathogens involved in polymicrobial sepsis and common clinical contaminants (Table 2). Melt curves were generated from standard dilutions reaching the digital level to ensure accurate curves for database creation. Calibrated and normalized helicity and difference curves of 12 clinically relevant bacteria were experimentally generated for the database (Figure 4). Highly pure and concentrated gDNA from laboratory stock organisms was used to generate standard melt curves, as any cross-contamination by other bacterial DNA would be amplified by the universal primers and contribute to changes in the melt curves. The amplicons were subsequently sequenced for identity validation (Supplementary Table 1). Difference curves of S. aureus, S. epidermidis and S. saprophyticus V6 amplicons were previously unresolvable (14), but under the currently optimized U-dHRM conditions are highly reproducible and discernible using only intercalating dye as the reporter (Figure 5). Next, a spiked mixture of S. aureus, E. faecalis and P. acnes was prepared, digitized, amplified by V6 broad-based primers and assessed by U-dHRM. Figure 6 contrasts a bulk HRMA experiment (red melt curve) and the U-dHRM results (all other melt curves) of the polymicrobial mixture. Input concentrations were adjusted such that the same amount of the polymicrobial gDNA was added to the bulk well as was distributed across the digital wells. Four curves were positively identified by matching to the database curves (Figure 6D). These were amplified from singular target templates. Knowing the number of negatives, 37, and total positives, 58, a Poisson calculation gives λoverall = 0.94 for the reaction mixture (Figure 6C). By this calculation, ∼31 of the unidentified 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 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/µl Taq, which gives a λPCR contaminants ∼ 0.5 for the polymicrobial U-dHRM assay. The original input mixture of S. aureus, E. faecalis and P. acnes gDNA then has λinput ∼ 0.44.Figure 4.

Bottom Line: The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR.We show that single-molecule detection and single nucleotide sensitivity is possible.U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA, Department of Emergency Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Infectious Disease, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA, Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University, Baltimore, MD 21218, USA, The Johns Hopkins Hospital, Baltimore, MD 21287, USA and Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.

ABSTRACT
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

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Related in: MedlinePlus