title: Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection.
abstract: High throughput automation is greatly enhanced using techniques that employ conveyor belt strategies with un-interrupted streams of flow. We have developed a 'conveyor belt' analog for high throughput real-time quantitative Polymerase Chain Reaction (qPCR) using droplet emulsion technology. We developed a low power, portable device that employs LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve multi-channel fluorescence detection for real-time fluorescence measurements. Continuously streaming fluid plugs or droplets pass through tubing wrapped around a two-temperature zone thermal block with each wrap of tubing fluorescently coupled to a 64-channel multi-anode PMT. This work demonstrates real-time qPCR of 0.1-10 μL droplets or fluid plugs over a range of 7 orders of magnitude concentration from 1 × 10(1) to 1 × 10(7). The real-time qPCR analysis allows dynamic range quantification as high as 1 × 10(7) copies per 10 μL reaction, with PCR efficiencies within the range of 90-110% based on serial dilution assays and a limit of detection of 10 copies per rxn. The combined functionality of continuous flow, low power thermal cycling, high throughput sample processing, and real-time qPCR improves the rates at which biological or environmental samples can be continuously sampled and analyzed.
authors.affiliation: Arizona State University School of Earth and Space Exploration, 781 E Terrace Road, ISTB4 Room 795, Tempe, AZ 85287, USA. Andrew.C.Hatch@asu.edu.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Hatch
authors.forename: Tathagata
authors.initials: T
authors.lastname: Ray
authors.forename: Kelly
authors.initials: K
authors.lastname: Lintecum
authors.forename: Cody
authors.initials: C
authors.lastname: Youngbull
mesh_terms: D001331:Automation; D004247:DNA / Q000032:analysis; D005336:Fiber Optic Technology; D005456:Fluorescent Dyes / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D004247:DNA