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Background  Routine culture-based diagnosis of  Pseudomonas aeruginosa  lung infection in Cystic Fibrosis (CF) patients can be hampered by the phenotypic variability of the microorganism, including its transition to a Viable But Non-Culturable (VBNC) state. The aim of this study was to validate an  ecfX- targeting qPCR protocol developed to detect all viable  P. aeruginosa  bacteria and to identify VBNC forms in CF sputum samples.    Methods  The study involved 115  P. aeruginosa  strains of different origins and 10 non- P. aeruginosa  strains and 88 CF sputum samples, 41 Culture-Positive (CP) and 47 Culture-Negative (CN). Spiking assays were performed using scalar dilutions of a mixture of live and dead  P. aeruginosa  ATCC 9027 and a pooled  P. aeruginosa -free sputum batch. Total DNA from sputum samples was extracted by a commercial kit, whereas a crude extract was obtained from the broth cultures. Extracellular DNA (eDNA) interference was evaluated by comparing the qPCR counts obtained from DNase-treated and untreated aliquots of the same samples. The statistical significance of the results was assessed by the Wilcoxon test and Student’s t test.    Results  The newly-developed qPCR protocol identified 96.6% of the  P. aeruginosa  isolates; no amplification was obtained with strains belonging to different species. Spiking assays supported protocol reliability, since counts always matched the amount of live bacteria, thus excluding the interference of dead cells and eDNA. The protocol sensitivity threshold was 70 cells/ml of the original sample. Moreover, qPCR detected  P. aeruginosa  in 9/47 CN samples and showed higher bacterial counts compared with the culture method in 10/41 CP samples.    Conclusions  Our findings demonstrate the reliability of the newly-developed qPCR protocol and further highlight the need for harnessing a non-culture approach to achieve an accurate microbiological diagnosis of  P. aeruginosa  CF lung infection and a greater understanding of its evolution.    Electronic supplementary material  The online version of this article (10.1186/s12879-018-3612-9) contains supplementary material, which is available to authorized users.

Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR: a validation study