g. a high cycle number indicates a low the initial concentration of P. aeruginosa in the sputum. Quality control of culture positive, PCR negative samples To exclude PCR inhibition as an explanation for the PCR negative, culture positive samples, the PCR mix, containing the DNA extract of the sample,
was spiked with an internal amplification control (IAC), as described by Khot et al. [14]. Briefly, 105 Jelly Fish oligonucleotides (105 bp) (IAC-oligo), 0.4 μM forward primer (IAC fw) and 0.4 μM reversed primer (IAC rev) primers were added to the reaction mix, and a separate qPCR experiment, using the SybrGreen kit, was carried out with primers hybridizing to the target DNA. When compared to a set of control samples, i.e. culture and qPCR P. aeruginosa
positive samples to which the HDAC inhibitor same amount of IAC had been Androgen Receptor Antagonist added, the PCR was considered as inhibited by (the DNA extract of) the sample, when an increase of 3 Cqs could be observed. To exclude that PCR negativity was due to primer mismatch with the oprL gene of the P. aeruginosa isolates for culture positive, PCR negative samples, oprL PCR was carried out on DNA extracted from the P. aeruginosa isolates, cultured from the same samples. Ethics The study was approved by the ethics committee from Ghent University Hospital (project nr. 2007/503). Written informed consent was obtained from the patients > 18 years, or from the parents for the children. Statistical analysis Differences in Cq values were examined using the Mann-Whitney U test and p values of < 0.05 were considered as significant.
Results A total of 852 samples was obtained from 397 not chronically infected CF patients, from six out of the seven Belgian cystic fibrosis centres. Of these, 729 samples (86%) from 307 patients remained P. aeruginosa negative by culture and by P. aeruginosa specific qPCR and 89 samples (10%) from 64 CF patients were both P. aeruginosa culture and qPCR positive (Additional File 1, Table S1). For 11 of the 89 samples (12%), only one culture method was positive, i.e. six times only MacConkey, five times only Cetrimide Broth. For these samples, the mean qPCR Cq-value was 28.6, while for the samples positive by both culture methods, the mean Cq value was 26.4 (Table 1) (p > 0.05, not significant). Table Buspirone HCl 1 Comparison of the sensitivity of detection by qPCR and culture Number of samples MacConkey Agar Cetrimide Broth qPCR Cq value (range, SD) 78 + + 26.4 (17-32, 4.3) 6 + – 29.8 (25-32, 2.7) 5 – + 27.3 (22-32, 4.3) 26 – - 31.7 (20-34, 3.2) 2 + – NA 3 – + NA 5 + + NA 729 – - NA NA: no amplification, SD: standard deviation Twenty-six samples (3%), obtained from 26 CF patients, were culture negative but qPCR positive (Additional File 1, Table S2). False positivity due to cross reaction with other CF associated bacterial species could be excluded because the PRIMA-1MET mw specificity of the primer set had been tested and confirmed on a broad set of common CF pathogenic species [13].