Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis

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.

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

“…An alternative, gene specific version LATE-PCR assay described elsewhere is also highly informative and very sensitive [42]. When looking to identify particular pathogens, the gene specific assay is capable of detecting down to a single genome copy of the gene even in the presence of other bacterial targets.…”

“…The Candida sp. and control primers and probes were the same as those used in the gene specific version of the assay [42]. Table 4 lists the bacterial and fungal genomic DNA targets of our pathogen test panel.…”

“…The 16S sepsis multiplex assay presented an alternative to the archetypal gene specific target approach [42]. The alternating conserved and highly variable regions of the 16S rDNA in bacteria made it possible to amplify the gene in many different species of bacteria while still differentiating between them.…”

“…One design used a combination of probes and primers specific to selected genes for each target to differentiate 17 pathogens of interest as well as an internal control [42]. The second design, described here, amplified just three targets, a bacterial 16S rDNA sequence, a sequence in the Candida P450L1A1 gene, and a sequence in Lactococcus lactis subsp.…”

Enhanced nucleic acid amplification with blood in situ by wire-guided droplet manipulation (WDM)

“…Rapid diagnostic tools to identify the cause of infection in blood are of extreme clinical importance due to the high mortality rates (49.7% overall for BSI) (Balk et al, 2001; Rice et al, 2013), significant morbidity, extreme cost of patient care (Kollef et al, 1999; Chakravorty et al, 2010; HCUP, 2009), and rising antimicrobial resistance rates (NNIS System, 2004). Rapid, point-of-care detection enables early use of targeted antibiotic therapy, to reduce morbidity and mortality, and timely isolation of colonized patients, to minimize transmission of antimicrobial resistance through the hospital setting (Akova et al, 2012).…”