Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity

Abstract

In this article we present a long target droplet polymerase chain reaction (PCR) microsystem for the amplification of the 16S ribosomal RNA gene. It is used for detecting Gram-positive and Gram-negative pathogens at high-throughput and is optimised for downstream species identification. The miniaturised device consists of three heating plates for denaturation, annealing and extension arranged to form a triangular prism. Around this prism a fluoropolymeric tubing is coiled, which represents the reactor. The source DNA was thermally isolated from bacterial cells without any purification, which proved the robustness of the system. Long target sequences up to 1.3 kbp from Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa have successfully been amplified, which is crucial for the successive species classification with DNA microarrays at high accuracy. In addition to the kilobase amplicon, detection limits down to DNA concentrations equivalent to 10(2) bacterial cells per reaction were achieved, which qualifies the microfluidic device for clinical applications. PCR efficiency could be increased up to 2-fold and the total processing time was accelerated 3-fold in comparison to a conventional thermocycler. Besides this speed-up, the device operates in continuous mode with consecutive droplets, offering a maximal throughput of 80 samples per hour in a single reactor. Therefore we have overcome the trade-off between target length, sensitivity and throughput, existing in present literature. This qualifies the device for the application in species identification by PCR and microarray technology with high sample numbers. Moreover early diagnosis of infectious diseases can be implemented, allowing immediate species specific antibiotic treatment. Finally this can improve patient convalescence significantly.

Screening and electrochemical detection of an antibiotic producing gene in bacteria on an integrated microchip

“…Such types of devices are rather far from miniaturization, and make the process tedious. 11 In contrast, the patterned ITO heater used in this work enables the realization of a handheld device on a monolithic chip. PDMS is also cost effective compared to other reports, in which a SU-8 based microchannel 12 or a silicon based device 13 was used.…”

The Dropletisation of Bio-Reactions

“…The PCR is one of the most practical molecular methods to amplify informative nucleic acid fragments especially from rare samples and single cells [12]. Stem cell and single cell research are gradually changing the clinical practice of stem cell therapy and molecular oncology.…”

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

“…Since its introduction (Kopp et al 1998), dynamic continuous-flow PCR devices have been realized by different structure designs and different fluidic geometries (Frey et al 2007;Ohashi et al 2007;Chien et al 2009;Hartung et al 2009;Pjescic et al 2010;Zhang and Xing 2010;Peham et al 2011). Among them, the oscillatoryflow PCR devices have obtained increasing attention in recent years, due to their salient features such as cycle number/dwell time flexibility, ease of real-time detection implementation, large footprint reduction, and ability to process multiple samples in parallel.…”