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Journal of Pathogens
Volume 2012 (2012), Article ID 424808, 10 pages
Research Article

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

1Department of Biology, Brandeis University, Waltham, MA 02453-2728, USA
2Smiths Detection Diagnostics, Edgewood, MD 21040, USA

Received 26 July 2012; Revised 2 October 2012; Accepted 12 October 2012

Academic Editor: Cormac G. M. Gahan

Copyright © 2012 Rachel K. Carver-Brown et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis.