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Future Microbiol. 2019 Jan;14:23-32. doi: 10.2217/fmb-2018-0197. Epub 2018 Dec 12.

Fast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR.

Author information

1
Research & Development, Abbott (Alere Technologies GmbH), Jena, Germany.
2
Center for Applied Research, InfectoGnostics Research Campus, Jena, Germany.
3
Institute for Infectious Diseases and Infection Control, University Medical Center of Jena, Jena, Germany.
4
Institute of Bacterial Infections & Zoonoses, Friedrich-Loeffler-Institute, Jena, Germany.
5
Institute for Medical Microbiology & Hygiene, Technical University of Dresden, Dresden, Germany.
6
Department for Optical Molecular Diagnostics and Systems Technology, Leibniz-Institute of Photonic Technology, Jena, Germany.

Abstract

AIM:

A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa).

MATERIALS & METHODS:

Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay.

RESULTS:

Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative.

CONCLUSION:

The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR.

KEYWORDS:

Gram-negative bacteria; TaqMan; colony PCR; molecular species determination; multicolor detection; multiplex; real-time PCR

PMID:
30539662
DOI:
10.2217/fmb-2018-0197
[Indexed for MEDLINE]
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