Journal: Science Advances
Article Title: Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief
Figure Lengend Snippet: Interfacial tension and fluorescence qPCR inhibition of the IE model. ( A ) Protein concentrations of the aortic, mitral, and tricuspid valve sections excised from a porcine heart and ground using a micro–mortar and pestle. The total protein concentration of the tissue model is 1.6 ± 0.1 mg/ml. ( B ) The interfacial tensions (γ) of clean and contaminated PCR mixtures are 25.55 and 27.60 mN/m, respectively. ( C ) Free-body force diagram with the interfacial layer. The forces on the droplet include the interfacial tension force ( F γ ), the buoyancy force ( F B ), the weight of the droplet ( F mg ), and the thermocouple force ( F TC ). ( D ) Fluorescence qPCR amplification curves for 16 S rRNA hypervariable regions V1-V2 and vanA gene from intact vancomycin-resistant E. faecium (VRE) with and without tissue contamination. The C t values for 16 S rRNA V1-V2 without tissue, 16 S rRNA V1-V2 with tissue, vanA without tissue, and vanA with tissue are 28.4, 30.0, 34.0, and 39.4, respectively. The tissue contamination inhibits fluorescence qPCR, as seen by the upward shift of 1.6 cycles for the 16 S rRNA V1-V2 target and 5.4 cycles for the vanA target. Additionally, NTC samples for each primer set are plotted. ( E ) Protein diffusion to the interface is calculated on the basis of typical blood and tissue concentrations, using diffusivities from literature and Fick’s equation. For comparison, the diffusion of Taq polymerase to the interface is also calculated. ( F and G ) The porcine heart from which heart valves were excised, sectioned, inoculated, ground, and used as the PCR target.
Article Snippet: Vancomycin-sensitive E. faecalis (VSE, ATCC 33186) and vancomycin-resistant E. faecium (VRE, Enterococcus ATCC 700221) were propagated, according to the procedure outlined in the American Type Culture Collection (ATCC) product sheet, to 108 CFU/ml, pelleted by centrifugation at 6000g for 10 min, resuspended in 100 μl of molecular grade water, and heat-killed at 95°C for 15 min. Purified K. pneumoniae strain Z026 genomic DNA was purchased from ZeptoMetrix.
Techniques: Fluorescence, Real-time Polymerase Chain Reaction, Inhibition, Protein Concentration, Polymerase Chain Reaction, Amplification, Diffusion-based Assay