Dnase rnase free water

This work was focused on the expression of the four major P. aeruginosa efflux pump genes (mexB, mexD, mexF and mexY). Normalization of expression results was carried out using rpsL (reference gene to normalize the relative amount of mRNA) and using the PA01 strain as a control. A LightCycler 96 (Roche Diagnostics, Meylan, France) was used for all quantitative PCRs. All PCR amplification reactions were performed in 96-well plates in a 10 μL final volume containing 2.5 μL of diluted (1:20) template cDNA, 1 μL of each primer (corresponding to a final concentration of 0.5 μM), 5 μL of QuantiTect SYBR Green PCR Master Mix (including MgCl₂ to reach a final concentration of 2.5 mM) (QIAGEN) and 0.5 μL of RNase/DNase free water (QIAGEN). The cycling program was set as follows: (1) activation: 1 cycle at 95°C for 15 min; (2) amplification: 45 cycles including a 15 s denaturation at 95°C, a 25 s annealing at 60°C and a 15 s elongation at 72°C; and (3) melting curve: 1 cycle including 5 s at 95°C, 1 min at 65°C and a final increase at 97°C with a transition rate of 0.11°C/s. Each reaction was carried out in duplicate and the experiment was repeated on two different sets of RNA extracts (biological replicate).^{4 ,7 (link),8}

All Chlamydiaceae-positive DNA extracts were included in further real-time PCR tests. Identification of C. psittaci based on the incA gene was conducted according to the protocol by Menard et al. (17 (link)), whereas C. gallinacea was detected by the amplification of an enoA gene fragment according to Laroucau et al. (14 (link)). Furthermore, specific real-time PCR assays were performed on selected samples to identify other Chlamydia species including C. abortus, C. pecorum, C. suis, and C. caviae (20 (link)), C. avium (39 (link)), and C. pneumoniae (12 (link)). In order to distinguish true target negatives from negatives due to PCR inhibition, an internal positive control (TaqMan Exogenous Internal Positive Control, Applied Biosystems, USA) was added to reaction mixtures according to the manufacturer’s instructions. Also, positive controls using C. abortus, C. pecorum, C. suis, and C. caviae, C. avium, and C. pneumoniae DNA, and negative equivalents using DNase-RNase free water (Qiagen, Germany) were run with each assay. All samples with a Ct value above 38.5 were considered negative.

All DNA extracts from birds (n = 1,830) were screened using a Chlamydiaceae-specific real-time PCR targeting a 23S rRNA gene fragment (111 bp) (4 (link)), which is conserved in all Chlamydiaceae. All analyses in the study were conducted on a 7500 Real-Time PCR System (Applied Biosystems, USA). Positive control using C. trachomatis (Genekam, Germany), and negative equivalents using DNase-RNase free water (Qiagen, Germany) were run with each assay. All sets of primers and probes used in this study are summarised in Supplementary file S3.