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

Content Provider	Springer Nature Link
Author	Peham, Johannes R. Grienauer, Walter Steiner, Hannes Heer, Rudolf Vellekoop, Michael J. Nöhammer, Christa Wiesinger Mayr, Herbert
Copyright Year	2011
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.
Starting Page	463
Ending Page	473
Page Count	11
File Format	PDF
ISSN	13872176
Journal	Biomedical Microdevices
Volume Number	13
Issue Number	3
e-ISSN	15728781
Language	English
Publisher	Springer US
Publisher Date	2011-01-27
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Long target High throughput Microfluidic Polymerase chain reaction Pathogen detection Bacteria Nanotechnology Engineering Fluid Dynamics Biophysics and Biological Physics Biomedical Engineering
Content Type	Text
Resource Type	Article
Subject	Nanoscience and Nanotechnology Molecular Biology Biomedical Engineering

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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