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Published on: July 9, 2012
Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis
1Department of Biology, Brandeis University, Waltham, MA 02453-2728, USA.
Aims:
The goal of this study was to construct a single-tube multiplex molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for the detection of 17 microbial pathogens commonly associated with septicaemia.
Methods And Results:
The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene-specific sequences. The pathogens and their targeted genes were: Klebsiella spp. (phoE); Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp. (thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf), Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified gene in Lactococcus lactis, served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence-specific low-temperature fluorescent probe derived from molecular beacons.
Conclusions:
All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA.
Significance And Impact Of The Study:
This assay used new technology to achieve an advance in the field of molecular diagnostics: a single-tube assay for detection of pathogens commonly responsible for septicaemia.
