Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection

Andrew C. Hatch, Tathagata Ray, Kelly Lintecum, Cody Youngbull

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 twoerature 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 × 101 to 1 × 107. The real-time qPCR analysis allows dynamic range quantification as high as 1 × 107 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.

Original languageEnglish (US)
Pages (from-to)562-568
Number of pages7
JournalLab on a Chip - Miniaturisation for Chemistry and Biology
Volume14
Issue number3
DOIs
StatePublished - Feb 7 2014

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Polymerase chain reaction
Real-Time Polymerase Chain Reaction
Fluorescence
Equipment and Supplies
Hot Temperature
Throughput
Tubing
Fluids
Automation
Thermal cycling
Emulsions
Dynamic analysis
Fiber optics
Dilution
Light emitting diodes
Limit of Detection
Assays
Anodes
Electrodes
Technology

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)
  • Bioengineering
  • Biomedical Engineering

Cite this

Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection. / Hatch, Andrew C.; Ray, Tathagata; Lintecum, Kelly; Youngbull, Cody.

In: Lab on a Chip - Miniaturisation for Chemistry and Biology, Vol. 14, No. 3, 07.02.2014, p. 562-568.

Research output: Contribution to journalArticle

Hatch, Andrew C. ; Ray, Tathagata ; Lintecum, Kelly ; Youngbull, Cody. / Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection. In: Lab on a Chip - Miniaturisation for Chemistry and Biology. 2014 ; Vol. 14, No. 3. pp. 562-568.
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