Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.
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.
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.
Stephanie I
SI
Fraley
Justin
J
Hardick
Billie J
BJ
Masek
Billie
B
Jo Masek
Pornpat
P
Athamanolap
Richard E
RE
Rothman
Charlotte A
CA
Gaydos
Karen C
KC
Carroll
Teresa
T
Wakefield
Tza-Huei
TH
Wang
Samuel
S
Yang
D000465:Algorithms; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D003936:Diagnosis, Computer-Assisted / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D018805:Sepsis / Q000175:diagnosis; D017421:Sequence Analysis
D035683:MicroRNAs