A Reusable Flow-Through Polymerase Chain Reaction Instrument for the Continuous Monitoring of Infectious Biological Agents

Abstract

Continuous monitoring of the environment for infectious diseases and related biowarfare agents requires the implementation of practical cost-effective methodologies that are highly sensitive and specific. One compatible method employed in clinical diagnostics is real-time polymerase chain reaction (PCR) analysis. The utility of this technique for environmental monitoring is limited, however, by the utilization of single-use consumables in commercial PCR instruments. This greatly increases mechanical complexity, because sophisticated robotic mechanisms must replenish the disposable elements. An alternative strategy develops an autonomous monitoring system consisting of reusable modules that readily interface with fluidic circuitry in a flow-through scheme. The reduced complexity should increase reliability while decreasing operating costs. In this report, we describe a reusable, flow-through PCR module that functions as one component in such a system. This module was rigorously evaluated with Bacillus anthracis genomic DNA and demonstrated high repeatability, sensitivity, and efficiency, with no evidence of sample-to-sample carryover.

On-Chip, Real-Time, Single-Copy Polymerase Chain Reaction in Picoliter Droplets

“…It quickly became the "gold standard" for effectiveness, specificity and sensitivity, and has been used almost exclusively for rapid characterization of pathogens because it does not require post-PCR manipulation and integration with capillary or gel electrophoresis, hybridization arrays, or mass spectrometry 2,3 . The next advances for rapid detection and characterization focused on reducing the thermal mass and heat diffusion distance into the aqueous sample to decrease PCR cycle time 2 .…”

The Autonomous Pathogen Detection System

“…Nucleic-acid detection was integrated using a Flow-Through PCR module based on earlier PCR devices developed at LLNL with DoD and DOE funding. 19,20 The flow-through format is ideally suited to the APDS's fluidics platform; the PCR module integrates essentially as a clamp-on reactor and detector on the existing valves and tubing. This next version of the instrument, dubbed the APDS150, used the multiplexed immunoassay as the primary test and real-time TaqMan PCR as a secondary, orthogonal test.…”

A Practical Liquid Plug Flow-through Polymerase Chain-Reaction System Based on a Heat-Resistant Resin Chip

“…In addition, this approach can be applied to reverse-transcription PCR (RT-PCR) by integrating the microfluidic RT process on a single chip, as demonstrated by the rapid diagnosis of influenza. [5][6][7] However, there are inevitable drawbacks resulting from destabilization of the fluid flow in the 95 C denaturation zone, which is near to the boiling point of water. In particular, the generation of air bubbles disrupts the microfluidic flow.…”