Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis
Article first published online: 20 DEC 2012
© 2012 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 114, Issue 2, pages 457–469, February 2013
How to Cite
Rice, L.M., Reis, A.H., Ronish, B., Carver-Brown, R.K., Czajka, J.W., Gentile, N., Kost, G. and Wangh, L.J. (2013), Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis. Journal of Applied Microbiology, 114: 457–469. doi: 10.1111/jam.12061
- Issue published online: 14 JAN 2013
- Article first published online: 20 DEC 2012
- Accepted manuscript online: 8 NOV 2012 11:01AM EST
- Manuscript Accepted: 30 OCT 2012
- Manuscript Revised: 2 OCT 2012
- Manuscript Received: 1 JUN 2012
- NIH. Grant Number: 1RC1EB010543-01 and 5RC1EB010643-02
- Smiths Detection Diagnostics, Inc.
- fluorescent contours;
- fluorescent signatures;
- sequence-specific low temperature probes
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.
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.