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

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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 102 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.

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Acknowledgement

The authors gratefully acknowledge S. Schönthaler for the expert technical assistance.

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Authors and Affiliations

  1. Health & Environment Department, AIT Austrian Institute of Technology, Muthgasse 11, 1190, Wien, Austria

    Johannes R. Peham, Rudolf Heer, Christa Nöhammer & Herbert Wiesinger-Mayr

  2. Aerospace and Advanced Composites GmbH, 2444, Seibersdorf, Austria

    Walter Grienauer & Hannes Steiner

  3. Institute of Sensor and Actuator Systems, Vienna University of Technology, Gußhausstrasse 27-29, 1040, Wien, Austria

    Michael J. Vellekoop

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  1. Johannes R. Peham
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  2. Walter Grienauer
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Correspondence to Johannes R. Peham.

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Peham, J.R., Grienauer, W., Steiner, H. et al. Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity. Biomed Microdevices 13, 463–473 (2011). https://doi.org/10.1007/s10544-011-9514-x

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