Abstract
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.
Original language | English (US) |
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Pages (from-to) | 457-469 |
Number of pages | 13 |
Journal | Journal of Applied Microbiology |
Volume | 114 |
Issue number | 2 |
DOIs | |
State | Published - Feb 2013 |
Keywords
- Endpoint
- Fluorescent contours
- Fluorescent signatures
- Linear-after-the-exponential-PCR
- Septicaemia
- Sequence-specific low temperature probes
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Biotechnology