Abstract

Rapid and species-specific detection, and virulence evaluation of opportunistic pathogens such as Pseudomonas aeruginosa, are issues that increasingly has attracted the attention of public health authorities. A set of primers and hydrolysis probe was designed based on one of the P. aeruginosa housekeeping genes, gyrB, and its specificity and sensitivity was evaluated by TaqMan qPCR methods. The end point PCR and SYBR Green qPCR were used as control methods. Furthermore, multiplex RT-qPCRs were developed for gyrB as reference and four virulence genes, including lasB, lasR, rhlR and toxA. Totally, 40 environmental samples, two clinical isolates from CF patients, two standard strains of P. aeruginosa, and 15 non-target reference strains were used to test the sensitivity and specificity of qPCR assays. In silico and in vitro cross-species testing confirmed the high specificity and low cross-species amplification of the designed gyrB418F/gyrB490R/gyrB444P. The sensitivity of both TaqMan and SYBR Green qPCRs was 100 for all target P. aeruginosa, and the detected count of bacteria was below ten genomic equivalents. The lowest M value obtained from gene-stability measurement was 0.19 that confirmed the suitability of gyrB as the reference gene for RT-qPCR. The developed qPCRs have enough detection power for identification of P. aeruginosa in environmental samples including clean and recreational water, treated and untreated sewage and soil. The short amplicon length of our designed primers and probes, alongside with a low M value, make it as a proper methodology for RT-qPCR in virulence genes expression assessment.