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Published on: October 23, 2011
Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical
N L Gentile1, A M Dillier, G V Williams
1Point-of-Care Testing Center for Teaching and Research (POCT•CTR), Pathology and Laboratory Medicine, School of Medicine, University of California, Davis, CA 95616, USA. nlgentile@ucdavis.edu
Aims:
To verify monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying 17 microbial pathogens (i.e., Klebsiella sp., Acinetobacter baumannii, Staphylococcus aureus, Enterobacter sp., Pseudomonas aeruginosa, coagulase negative staphylococci, Enterococcus sp., Candida sp.) commonly associated with septicaemia using clinical isolates.
Methods And Results:
Clinical isolates of each target pathogen were collected from the University of California, Davis Medical Center (UCDMC) microbiology laboratory. Five microlitres (μl) of each culture suspension (1 × 10(8) CFU ml(-1) ) were added to 20 μl of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except Enterobacter cloacae (2.7%), Ac. baumannii (57%) and Ps. aeruginosa (97.8%). All clinical isolates were positive in multiplex, with the exception of two Ac. baumannii, two Klebsiella oxytoca and two Candida parapsilosis isolates.
Conclusions:
Sixteen pathogens can be identified by monoplex LATE-PCR assays with sensitivities ≥ 97.8%. The multiplex assay demonstrated 91.4% sensitivity when tested with DNA extracted from 70 different target strains.
Significance And Impact Of The Study:
This study demonstrates the potential of LATE-PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.
Insights
This study verified monoplex and multiplex linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying common septicaemia pathogens. The assays show potential as a culture adjunct, with monoplex assays achieving high sensitivity for most pathogens.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Septicaemia is a life-threatening condition caused by microbial pathogens.
- Accurate and rapid identification of causative agents is crucial for effective treatment.
- Current diagnostic methods, primarily culture-based, can be time-consuming.
Purpose of the Study:
- To evaluate the performance of monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays.
- To identify 17 common microbial pathogens associated with septicaemia using clinical isolates.
- To assess the potential of LATE-PCR as an adjunct to traditional culture methods.
Main Methods:
- Clinical isolates of target pathogens were obtained from a medical center microbiology laboratory.
- Monoplex LATE-PCR assays were performed using a standard input level of microbial culture suspension.
- Multiplex LATE-PCR assays were conducted using DNA extracted from clinical isolates.
Main Results:
- Monoplex assays showed 100% sensitivity for most pathogens at the tested input level, with exceptions for Enterobacter cloacae (2.7%), Acinetobacter baumannii (57%), and Pseudomonas aeruginosa (97.8%).
- The multiplex assay identified all tested clinical isolates, with minor exceptions for two isolates each of Acinetobacter baumannii, Klebsiella oxytoca, and Candida parapsilosis.
- Overall, monoplex assays identified 16 pathogens with sensitivities of 97.8% or higher, while the multiplex assay achieved 91.4% sensitivity with extracted DNA.
Conclusions:
- Monoplex LATE-PCR assays are highly sensitive for identifying most common septicaemia pathogens.
- The multiplex LATE-PCR assay demonstrates good performance for identifying a broad range of pathogens from clinical samples.
- LATE-PCR holds promise as a rapid diagnostic tool to complement existing culture-based methods for septicaemia detection, pending further optimization and validation.
