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.