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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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U-dHRM identifies bacteria in a clinical blood bottle. (a) Standard database melt curves generated from four types of bacteria isolated from the polymicrobial blood bottle. (b) Normalized, temperature calibrated and database matched digital melt curves after 70 cycles of PCR. Broad-based amplified, single copy detection of E. faecalis in the blood sample extract was achieved (brown melt curve). The number of wells containing single contaminating gDNA templates from Taq polymerase (light gray non-matching curves) is few. Negative wells (dark gray melt curves) give a calculated λ = 0.075. The number of copies detected is shown in parentheses next to each legend label. The corresponding ‘bulk’ melt curve (red curve) where an amount of DNA sample equivalent to that diluted across the rest of the plate, and also including contaminating Taq gDNA, was assayed by conventional bulk HRMA.
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gkt684-F7: U-dHRM identifies bacteria in a clinical blood bottle. (a) Standard database melt curves generated from four types of bacteria isolated from the polymicrobial blood bottle. (b) Normalized, temperature calibrated and database matched digital melt curves after 70 cycles of PCR. Broad-based amplified, single copy detection of E. faecalis in the blood sample extract was achieved (brown melt curve). The number of wells containing single contaminating gDNA templates from Taq polymerase (light gray non-matching curves) is few. Negative wells (dark gray melt curves) give a calculated λ = 0.075. The number of copies detected is shown in parentheses next to each legend label. The corresponding ‘bulk’ melt curve (red curve) where an amount of DNA sample equivalent to that diluted across the rest of the plate, and also including contaminating Taq gDNA, was assayed by conventional bulk HRMA.

Mentions: As further validation of the utility of U-dHRM, we next tested a clinical blood bottle from a patient identified as having polymicrobial infection with E. faecalis, Enterococcus faecium, Aeromonas caviae and K. pneumoniae. For these experiments, we relied on the Johns Hopkins Hospital clinical microbiology laboratory for culture and phenotypic identification of the bacteria in the sample as the gold standard reference. Because our database does not yet include melt curves for all clinically relevant bacteria, which numbers in the hundreds, we also acquired the blood bottle bacteria as isolates from the clinical microbiology laboratory to generate additional database curves (Figure 7A). DNA extracted from the blood bottle was then tested with U-dHRM and compared with the database organisms for matching. Figure 7B shows the resulting melt curves where E. faecalis was correctly identified at the single cell level. The bulk melt curve of the blood DNA extract did not match curves in the database. Reaction volume reduction to 1 µl reduced the number of non-matching contaminating DNA such that an overall λ = 0.075 was achieved. Technology that greatly increases the number of digital reactions, decreases the reaction volume and incorporates U-dHRM is needed to achieve absolute quantitation of each bacterial species (see ‘Discussion’ section).Figure 7.


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)

U-dHRM identifies bacteria in a clinical blood bottle. (a) Standard database melt curves generated from four types of bacteria isolated from the polymicrobial blood bottle. (b) Normalized, temperature calibrated and database matched digital melt curves after 70 cycles of PCR. Broad-based amplified, single copy detection of E. faecalis in the blood sample extract was achieved (brown melt curve). The number of wells containing single contaminating gDNA templates from Taq polymerase (light gray non-matching curves) is few. Negative wells (dark gray melt curves) give a calculated λ = 0.075. The number of copies detected is shown in parentheses next to each legend label. The corresponding ‘bulk’ melt curve (red curve) where an amount of DNA sample equivalent to that diluted across the rest of the plate, and also including contaminating Taq gDNA, was assayed by conventional bulk HRMA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt684-F7: U-dHRM identifies bacteria in a clinical blood bottle. (a) Standard database melt curves generated from four types of bacteria isolated from the polymicrobial blood bottle. (b) Normalized, temperature calibrated and database matched digital melt curves after 70 cycles of PCR. Broad-based amplified, single copy detection of E. faecalis in the blood sample extract was achieved (brown melt curve). The number of wells containing single contaminating gDNA templates from Taq polymerase (light gray non-matching curves) is few. Negative wells (dark gray melt curves) give a calculated λ = 0.075. The number of copies detected is shown in parentheses next to each legend label. The corresponding ‘bulk’ melt curve (red curve) where an amount of DNA sample equivalent to that diluted across the rest of the plate, and also including contaminating Taq gDNA, was assayed by conventional bulk HRMA.
Mentions: As further validation of the utility of U-dHRM, we next tested a clinical blood bottle from a patient identified as having polymicrobial infection with E. faecalis, Enterococcus faecium, Aeromonas caviae and K. pneumoniae. For these experiments, we relied on the Johns Hopkins Hospital clinical microbiology laboratory for culture and phenotypic identification of the bacteria in the sample as the gold standard reference. Because our database does not yet include melt curves for all clinically relevant bacteria, which numbers in the hundreds, we also acquired the blood bottle bacteria as isolates from the clinical microbiology laboratory to generate additional database curves (Figure 7A). DNA extracted from the blood bottle was then tested with U-dHRM and compared with the database organisms for matching. Figure 7B shows the resulting melt curves where E. faecalis was correctly identified at the single cell level. The bulk melt curve of the blood DNA extract did not match curves in the database. Reaction volume reduction to 1 µl reduced the number of non-matching contaminating DNA such that an overall λ = 0.075 was achieved. Technology that greatly increases the number of digital reactions, decreases the reaction volume and incorporates U-dHRM is needed to achieve absolute quantitation of each bacterial species (see ‘Discussion’ section).Figure 7.

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.

Show MeSH
Related in: MedlinePlus