Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates

N. L. Gentile, A. M. Dillier, G. V. Williams, J. Ackers, A. H. Reis, L. M. Rice, L. J. Wangh, J. W. Czajka, Gerald J Kost

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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 × 108 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.

Original languageEnglish (US)
Pages (from-to)586-594
Number of pages9
JournalJournal of Applied Microbiology
Volume114
Issue number2
DOIs
StatePublished - Feb 2013

Fingerprint

Acinetobacter baumannii
Sepsis
Candida
Polymerase Chain Reaction
Pseudomonas aeruginosa
Klebsiella oxytoca
Genes
Enterobacter cloacae
Enterobacter
Klebsiella
Coagulase
DNA
Enterococcus
Microbiology
Staphylococcus
Staphylococcus aureus
Suspensions

Keywords

  • Detection
  • Diseases
  • Identification
  • Polymerase chain reaction
  • Virulence

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Biotechnology

Cite this

Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates. / Gentile, N. L.; Dillier, A. M.; Williams, G. V.; Ackers, J.; Reis, A. H.; Rice, L. M.; Wangh, L. J.; Czajka, J. W.; Kost, Gerald J.

In: Journal of Applied Microbiology, Vol. 114, No. 2, 02.2013, p. 586-594.

Research output: Contribution to journalArticle

Gentile, NL, Dillier, AM, Williams, GV, Ackers, J, Reis, AH, Rice, LM, Wangh, LJ, Czajka, JW & Kost, GJ 2013, 'Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates', Journal of Applied Microbiology, vol. 114, no. 2, pp. 586-594. https://doi.org/10.1111/jam.12062
Gentile, N. L. ; Dillier, A. M. ; Williams, G. V. ; Ackers, J. ; Reis, A. H. ; Rice, L. M. ; Wangh, L. J. ; Czajka, J. W. ; Kost, Gerald J. / Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates. In: Journal of Applied Microbiology. 2013 ; Vol. 114, No. 2. pp. 586-594.
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AU - Williams, G. V.

AU - Ackers, J.

AU - Reis, A. H.

AU - Rice, L. M.

AU - Wangh, L. J.

AU - Czajka, J. W.

AU - Kost, Gerald J

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N2 - 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 × 108 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.

AB - 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 × 108 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.

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